Combination therapies for cancer

ABSTRACT

A novel combination comprising a B-Raf inhibitor, particularly N-{3-[5-(2-Amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide or a pharmaceutically acceptable salt thereof, the MEK inhibitor N-{3-[3-cyclopropyl-5-(2-fluoro-4-iodo-phenylamino)6,8-dimethyl; -2,4,7-trioxo-3,4,6,7-tetrahydro-2H-pyrido[4,3-d]pyrimidin-1-yl]phenyl}acetamide, or a pharmaceutically acceptable salt or solvate thereof, and a PD-1 antagonist; pharmaceutical compositions comprising the same and methods of using such combinations and compositions in the treatment of conditions in which the inhibition of MEK and/or B-Raf and/or immune modulation through PD-1 is beneficial, e.g., cancer.

FIELD OF THE INVENTION

The present invention relates to a method of treating cancer in a mammaland to combinations useful in such treatment. In particular, the methodrelates to a novel combination comprising a B-Raf inhibitor,particularlyN-{3-[5-(2-Amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamideor a pharmaceutically acceptable salt thereof, and/or the MEK inhibitorN-{3-[3-cyclopropyl-5-(2-fluoro-4-iodo-phenylamino)6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydro-2H-pyrido[4,3-d]pyrimidin-1-yl]phenyl}acetamide,or a pharmaceutically acceptable salt or solvate thereof, and a PD-1antagonist (e.g., an anti-PD-1 antibody or antigen binding fragmentthereof, or an anti-PD-Ligand antibody or antigen binding fragmentthereof); pharmaceutical compositions comprising the same and methods ofusing such combinations and compositions in the treatment of conditionsin which the inhibition of MEK and/or inhibition of B-Raf and/orinhibiting endogenous PD-L1 and/or PD-L2 from binding PD-1 isbeneficial, e.g., cancer.

BACKGROUND OF THE INVENTION

Effective treatment of hyperproliferative disorders including cancer isa continuing goal in the oncology field. Generally, cancer results fromthe deregulation of the normal processes that control cell division,differentiation and apoptotic cell death and is characterized by theproliferation of malignant cells which have the potential for unlimitedgrowth, local expansion and systemic metastasis. Deregulation of normalprocesses include abnormalities in signal transduction pathways andresponse to factors which differ from those found in normal cells.

An important large family of enzymes is the protein kinase enzymefamily. Currently, there are about 500 different known protein kinases.Protein kinases serve to catalyze the phosphorylation of an amino acidside chain in various proteins by the transfer of the γ-phosphate of theATP-Mg²⁺ complex to said amino acid side chain. These enzymes controlthe majority of the signaling processes inside cells, thereby governingcell function, growth, differentiation and destruction (apoptosis)through reversible phosphorylation of the hydroxyl groups of serine,threonine and tyrosine residues in proteins.

Studies have shown that protein kinases are key regulators of many cellfunctions, including signal transduction, transcriptional regulation,cell motility, and cell division. Several oncogenes have also been shownto encode protein kinases, suggesting that kinases play a role inoncogenesis. These processes are highly regulated, often by complexintermeshed pathways where each kinase will itself be regulated by oneor more kinases. Consequently, aberrant or inappropriate protein kinaseactivity can contribute to the rise of disease states associated withsuch aberrant kinase activity including benign and malignantproliferative disorders as well as diseases resulting from inappropriateactivation of the immune and nervous systems. Due to their physiologicalrelevance, variety and ubiquitousness, protein kinases have become oneof the most important and widely studied family of enzymes inbiochemical and medical research.

The protein kinase family of enzymes is typically classified into twomain subfamilies: Protein Tyrosine Kinases and Protein Serine/ThreonineKinases, based on the amino acid residue they phosphorylate. The proteinserine/threonine kinases (PSTK), includes cyclic AMP- and cyclicGMP-dependent protein kinases, calcium and phospholipid dependentprotein kinase, calcium- and calmodulin-dependent protein kinases,casein kinases, cell division cycle protein kinases and others. Thesekinases are usually cytoplasmic or associated with the particulatefractions of cells, possibly by anchoring proteins. Aberrant proteinserine/threonine kinase activity has been implicated or is suspected ina number of pathologies such as rheumatoid arthritis, psoriasis, septicshock, bone loss, many cancers and other proliferative diseases.Accordingly, serine/threonine kinases and the signal transductionpathways which they are part of are important targets for drug design.The tyrosine kinases phosphorylate tyrosine residues. Tyrosine kinasesplay an equally important role in cell regulation. These kinases includeseveral receptors for molecules such as growth factors and hormones,including epidermal growth factor receptor, insulin receptor, plateletderived growth factor receptor and others. Studies have indicated thatmany tyrosine kinases are transmembrane proteins with their receptordomains located on the outside of the cell and their kinase domains onthe inside. Much work is also in progress to identify modulators oftyrosine kinases as well.

Receptor tyrosine kinases (RTKs) catalyze phosphorylation of certaintyrosyl amino acid residues in various proteins, including themselves,which govern cell growth, proliferation and differentiation.

Downstream of the several RTKs lie several signaling pathways, amongthem is the Ras-Raf-MEK-ERK kinase pathway. It is currently understoodthat activation of Ras GTPase proteins in response to growth factors,hormones, cytokines, etc. stimulates phosphorylation and activation ofRaf kinases. These kinases then phosphorylate and activate theintracellular protein kinases MEK1 and MEK2, which in turn phosphorylateand activate other protein kinases, ERK1 and 2. This signaling pathway,also known as the mitogen-activated protein kinase (MAPK) pathway orcytoplasmic cascade, mediates cellular responses to growth signals. Theultimate function of this pathway is to link receptor activity at thecell membrane with modification of cytoplasmic or nuclear targets thatgovern cell proliferation, differentiation, and survival.

The constitutive activation of this pathway is sufficient to inducecellular transformation. Disregulated activation of the MAP kinasepathway, due to aberrant receptor tyrosine kinase activation, Rasmutations or Raf mutations, has frequently been found in human cancers,and represents a major factor determining abnormal growth control. Inhuman malignances, Ras mutations are common, having been identified inabout 30% of cancers. The Ras family of GTPase proteins (proteins whichconvert guanosine triphosphate to guanosine diphosphate) relay signalsfrom activated growth factor receptors to downstream intracellularpartners. Prominent among the targets recruited by active membrane-boundRas are the Raf family of serine/threonine protein kinases. The Raffamily is composed of three related kinases (A-, B- and C-Raf) that actas downstream effectors of Ras. Ras-mediated Raf activation in turntriggers activation of MEK1 and MEK2 (MAP/ERK kinases 1 and 2), which inturn phosphorylate ERK1 and ERK2 (extracellular signal-regulated kinases1 and 2) on tyrosine-185 and threonine-183. Activated ERK1 and ERK2translocate and accumulate in the nucleus, where they can phosphorylatea variety of substrates, including transcription factors that controlcellular growth and survival. Given the importance of theRas/Raf/MEK/ERK pathway in the development of human cancers, the kinasecomponents of the signaling cascade are merging as potentially importanttargets for the modulation of disease progression in cancer and otherproliferative diseases.

MEK1 and MEK2 are members of a larger family of dual-specificity kinases(MEK1-7) that phosphorylate threonine and tyrosine residues of variousMAP kinases. MEK1 and MEK2 are encoded by distinct genes, but they sharehigh homology (80%) both within the C-terminal catalytic kinase domainsand the most of the N-terminal regulatory region. Oncogenic forms ofMEK1 and MEK2 have not been found in human cancers, but constitutiveactivation of MEK has been shown to result in cellular transformation.In addition to Raf, MEK can also be activated by other oncogenes aswell. So far, the only known substrates of MEK1 and MEK2 are ERK1 andERK2. This unusual substrate specificity in addition to the uniqueability to phosphorylate both tyrosine and threonine residues placesMEK1 and MEK2 at a critical point in the signal transduction cascadewhich allows these MEK proteins to integrate many extracellular signalsinto the MAPK pathway.

Accordingly, it has been recognized that an inhibitor of a protein ofthe MAPK kinase pathway (eg. MEK) should be of value both as ananti-proliferative, pro-apoptotic and anti-invasive agent for use in thecontainment and/or treatment of proliferative or invasive disease.

Moreover, it is also known that a compound having MEK inhibitoryactivity effectively induces inhibition of ERK1/2 activity andsuppression of cell proliferation (The Journal of Biological Chemistry,vol. 276, No. 4 pp. 2686-2692, 2001), and the compound is expected toshow effects on diseases caused by undesirable cell proliferation, suchas tumor genesis and/or cancer. Mutations in various Ras GTPases and theB-Raf kinase have been identified that can lead to sustained andconstitutive activation of the MAPK pathway, ultimately resulting inincreased cell division and survival. These mutations have been stronglylinked with the establishment, development, and progression of a widerange of human cancers. For example, in melanoma, more than 80% of theBRAF mutations cause a substitution of the amino acid glutamate (E) forvaline (V) at position 600 (V600E) of the BRAF protein, whereasapproximately 3-20% of melanoma mutations are a substitution of lysine(K) for valine at position 600 (V600K) (Gorden et al., Cancer Res (2003)63:3955-3957; Houben et al., J Carcinog (2004) 3:6; Kumar et al., ClinCancer Res. (2003) 9:3362-3368; Libra et al., Cell Cycle (2005)4:1382-1384; Omholt et al. Clin Cancer Res (2003) 9:6483-6488. Thebiological role of the Raf kinases, and specifically that of B-Raf, insignal transduction is described in Davies, H., et al., Nature (2002)9:1-6; Garnett, M. J. & Marais, R., Cancer Cell (2004) 6:313-319;Zebisch, A. & Troppmair, J., Cell. Mol. Life Sci. (2006) 63:1314-1330;Midgley, R. S. & Kerr, D. J., Crit. Rev. Onc/Hematol. (2002) 44:109-120;Smith, R. A., et al., Curr. Top. Med. Chem. (2006) 6:1071-1089; andDownward, J., Nat. Rev. Cancer (2003) 3:11-22.

Naturally occurring mutations of the B-Raf kinase that activate MAPKpathway signaling have been found in a large percentage of humanmelanomas (Davies (2002) supra) and thyroid cancers (Cohen et al J. Nat.Cancer Inst. (2003) 95(8) 625-627 and Kimura et al Cancer Res. (2003)63(7) 1454-1457), as well as at lower, but still significant,frequencies in the following:

Barret's adenocarcinoma (Garnett et al., Cancer Cell (2004) 6 313-319and Sommerer et al Oncogene (2004) 23(2) 554-558), billiary tractcarcinomas (Zebisch et al., Cell. Mol. Life Sci. (2006) 63 1314-1330),breast cancer (Davies (2002) supra), cervical cancer (Moreno-Bueno et alClin. Cancer Res. (2006) 12(12) 3865-3866), cholangiocarcinoma(Tannapfel et al Gut (2003) 52(5) 706-712), central nervous systemtumors including primary CNS tumors such as glioblastomas, astrocytomasand ependymomas (Knobbe et al Acta Neuropathol. (Berl.) (2004) 108(6)467-470, Davies (2002) supra, and Garnett et al., Cancer Cell (2004)supra) and secondary CNS tumors (i.e., metastases to the central nervoussystem of tumors originating outside of the central nervous system),colorectal cancer, including large intestinal colon carcinoma (Yuen etal Cancer Res. (2002) 62(22) 6451-6455, Davies (2002) supra and Zebischet al., Cell. Mol. Life Sci. (2006), gastric cancer (Lee et al Oncogene(2003) 22(44) 6942-6945), carcinoma of the head and neck includingsquamous cell carcinoma of the head and neck (Cohen et al J. Nat. CancerInst. (2003) 95(8) 625-627 and Weber et al Oncogene (2003) 22(30)4757-4759), hematologic cancers including leukemias (Garnett et al.,Cancer Cell (2004) supra, particularly acute lymphoblastic leukemia(Garnett et al., Cancer Cell (2004) supra and Gustafsson et al Leukemia(2005) 19(2) 310-312), acute myelogenous leukemia (AML) (Lee et alLeukemia (2004) 18(1) 170-172, and Christiansen et al Leukemia (2005)19(12) 2232-2240), myelodysplastic syndromes (Christiansen et alLeukemia (2005) supra) and chronic myelogenous leukemia (Mizuchi et alBiochem. Biophys. Res. Commun. (2005) 326(3) 645-651); Hodgkin'slymphoma (Figl et al Arch. Dermatol. (2007) 143(4) 495-499),non-Hodgkin's lymphoma (Lee et al Br. J. Cancer (2003) 89(10)1958-1960), megakaryoblastic leukemia (Eychene et al Oncogene (1995)10(6) 1159-1165) and multiple myeloma (Ng et al Br. J. Haematol. (2003)123(4) 637-645), hepatocellular carcinoma (Garnett et al., Cancer Cell(2004), lung cancer (Brose et al Cancer Res. (2002) 62(23) 6997-7000,Cohen et al J. Nat. Cancer Inst. (2003) supra and Davies (2002) supra),including small cell lung cancer (Pardo et al EMBO J. (2006) 25(13)3078-3088) and non-small cell lung cancer (Davies (2002) supra), ovariancancer (Russell & McCluggage J. Pathol. (2004) 203(2) 617-619 and Davies(2002) supr), endometrial cancer (Garnett et al., Cancer Cell (2004)supra, and Moreno-Bueno et al Clin. Cancer Res. (2006) supra),pancreatic cancer (Ishimura et al Cancer Lett. (2003) 199(2) 169-173),pituitary adenoma (De Martino et al J. Endocrinol. Invest. (2007) 30(1)RC1-3), prostate cancer (Cho et al Int. J. Cancer (2006) 119(8)1858-1862), renal cancer (Nagy et al Int. J. Cancer (2003) 106(6)980-981), sarcoma (Davies (2002) supra), and skin cancers(Rodriguez-Viciana et al Science (2006) 311(5765) 1287-1290 and Davies(2002) supra). Overexpression of c-Raf has been linked to AML (Zebischet al., Cancer Res. (2006) 66(7) 3401-3408, and Zebisch (Cell. Mol. LifeSci. (2006)) and erythroleukemia (Zebisch et al., Cell. Mol. Life Sci.(2006).

By virtue of the role played by the Raf family kinases in these cancersand exploratory studies with a range of preclinical and therapeuticagents, including one selectively targeted to inhibition of B-Raf kinaseactivity (King A. J., et al., (2006) Cancer Res. 66:11100-11105), it isgenerally accepted that inhibitors of one or more Raf family kinaseswill be useful for the treatment of such cancers or other conditionassociated with Raf kinase.

Mutation of B-Raf has also been implicated in other conditions,including cardio-facio cutaneous syndrome (Rodriguez-Viciana et alScience (2006) 311(5765) 1287-1290) and polycystic kidney disease (Nagaoet al Kidney Int. (2003) 63(2) 427-437).

Programmed Cell Death 1 (PD-1) is a 50-55 kDa type I transmembranereceptor originally identified by subtractive hybridization of a mouse Tcell line undergoing apoptosis (Ishida et al., 1992, Embo J.11:3887-95). A member of the CD28 gene family, PD-1 is expressed onactivated T, B, and myeloid lineage cells (Greenwald et al., 2005, Annu.Rev. Immunol. 23:515-48; Sharpe et al., 2007, Nat. Immunol. 8:239-45).

U.S. Pat. Nos. 6,808,710 and 7,101,550, issued to C. Wood and G. Freemanon Oct. 26, 2004 and Sep. 5, 2006, respectively, disclose methods forattempting to modulate an immune response by activating or inhibitingsignaling of the PD-1 receptor using, for example, an antibody thatbinds PD-1.

Based on the observation that blocking PD-1 inhibitory signals at timeof priming decreases immune cell responsiveness, U.S. Pat. No.7,029,674, issued Apr. 18, 2006 to B. Carreno and J. Leonard, disclosesmethods to decrease activation of an immune cell by contacting the cellwith an agent that inhibits PD-1 signaling. Additionally, U.S. Pat. No.7,595,048 and U.S. Pat. No. 8,168,179 disclose methods of treatingcancer with anti-PD-1 antibodies.

The PD-1 pathway has been of considerable interest to researchersdeveloping therapies to treat melanoma and other tumor types. The PD-1receptor is expressed on the surface of activated T cells and binds toligands on the surface of antigen-presenting cells (PD-L1 and PD-L2), aninteraction that modulates immune response. Many cancer cells expresshigh levels of PD-L1 on their surface, which cause T cells to switch offthrough PD-L1's interaction with PD-1, rendering them unable to generatean antitumor response.

PD-1 negatively modulates T cell activation, and this inhibitoryfunction is linked to an immunoreceptor tyrosine-based inhibitory motif(ITIM) of its cytoplasmic domain (Greenwald et al., supra; Parry et al.,2005, Mol. Cell. Biol. 25:9543-53). Disruption of this inhibitoryfunction of PD-1 can lead to autoimmunity. For example, PD-1 knockout inC57B1/6 mice leads to a lupus-like syndrome, whereas in BALB/c mice itleads to development of dilated cardiomyopathy (Nishimura et al., 1999,Immunity 11:141-51; Okazaki et al., 2003, Nat. Med. 9:1477-83). Inhumans, a single nucleotide polymorphism in PD-1 gene locus isassociated with higher incidences of systemic lupus erythematosus, type1 diabetes, rheumatoid arthritis, and progression of multiple sclerosis.The reverse scenario can also be deleterious. Sustained negative signalsby PD-1 have been implicated in T cell dysfunctions in many pathologicsituations, such as tumor immune evasion and chronic viral infections.

Host anti-tumor immunity is mainly affected by tumor-infiltratinglymphocytes (TILs) (Galore et al., 2006, Science 313:1960-4). Multiplelines of evidence have indicated that TILs are subject to PD-1inhibitory regulation. First, PD-L1 expression is confirmed in manyhuman and mouse tumor lines and the expression can be furtherupregulated by IFN-.gamma in vitro (Dong et al., 2002, Nat. Med.8:793-800). Second, expression of PD-L1 by tumor cells has been directlyassociated with their resistance to lysis by anti-tumor T cells in vitro(Dong et al., supra; Blank et al., 2004, Cancer Res. 64:1140-5). Third,PD-1 knockout mice are resistant to tumor challenge (Iwai et al., 2005,Int. Immunol. 17:133-44) and T cells from PD-1 knockout mice are highlyeffective in tumor rejection when adoptively transferred totumor-bearing mice (Blank et al., supra). Fourth, blocking PD-1inhibitory signals by a monoclonal antibody can potentiate hostanti-tumor immunity in mice (Iwai et al., supra; Hirano et al., 2005,Cancer Res. 65:1089-96). Fifth, high degrees of PD-L1 expression intumors (detected by immunohistochemical staining) are associated withpoor prognosis for many human cancer types (Hamanishi et al., 2007,Proc. Natl. Acad. Sci. USA 104:3360-5).

Though there have been many recent advances in the treatment of cancer,there remains a need for more effective and/or enhanced treatment of anindividual suffering the effects of cancer. The current inventionaddresses this need.

SUMMARY OF THE INVENTION

The current invention is directed to a combination of a B-Raf inhibitor,and/or a MEK inhibitor, and an anti-PD-1 antibody or antigen bindingfragment thereof in the treatment of cancer.

The present invention is directed to a combination of therapeutic agentsthat is advantageous over treatment with each agent when administeredalone and advantageous over treatment with a combination of a MEKinhibitor and a B-RAF inhibitor. In particular, the drug combinationthat includes the B-Raf inhibitorN-{3-[5-(2-Amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamideor a pharmaceutically acceptable salt thereof, and/or the MEK inhibitorN-{3-[3-cyclopropyl-5-(2-fluoro-4-iodo-phenylamino)6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydro-2H-pyrido[4,3-d]pyrimidin-1-yl]phenyl}acetamide,or a pharmaceutically acceptable salt or solvate thereof, and a PD-1antagonist (e.g., MK-3475 or an antigen binding fragment thereof) isdescribed.

The MEK inhibitor of the invention is represented by the structure ofstructure (I):

or a pharmaceutically acceptable salt or solvate thereof (collectivelyreferred to herein as “Compound A”),

The B-Raf inhibitor of the invention is represented by the structure ofstructure (II):

or a pharmaceutically acceptable salt thereof (collectively referred toherein as “Compound B”).

The PD-1 antagonist of the invention inhibits the binding of PD-L1 toPD-1, and preferably also inhibits the binding of PD-L2 to PD-1.Preferably, the PD-1 antagonist is a monoclonal antibody, or an antigenbinding fragment thereof, which specifically binds to PD-1 and blocksthe binding of PD-L1 to PD-1. In one particularly preferred embodiment,the PD-1 antagonist is an anti-PD-1 monoclonal antibody that binds toPD-1 and blocks binding of both PD-L1 and PD-L2 to PD-1.

Nivolumab, which is also known as BMS-936558 and MDX1106, is a fullyhuman IgG4 monoclonal antibody which binds to PD-1 and is designed forthe treatment of cancer by Bristol-Myers Squibb and Ono Pharmaceuticals.

MK-3475, which was previously known as lambrolizumab, is a humanizedIgG4 monoclonal antibody which binds to PD-1 and is being developed byMerck and Co for the treatment of cancer.

In a first aspect of the present invention, there is provided acombination comprising a PD-1 antagonist and one or both of a Compound Aand a Compound B, wherein:

the Compound B is a compound of structure (II)

or a pharmaceutically acceptable salt thereof; and

the Compound A is a compound of structure (I):

or a pharmaceutically acceptable salt or solvate thereof.

In another aspect of the invention, there is provided a combinationcomprising a PD-1 antagonist and one or both of a Compound A and aCompound B, wherein the Compound B isN-{3-[5-(2-Amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamidemethanesulfonate; and the Compound A isN-{3-[3-cyclopropyl-5-(2-fluoro-4-iodo-phenylamino)6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydro-2H-pyrido[4,3-d]pyrimidin-1-yl]phenyl}acetamidedimethyl sulfoxide (solvate).

In another aspect of the present invention, there is provided acombination, comprising a PD-1 antagonist and one or both of a CompoundA and a Compound B for use in therapy, wherein:

the Compound B is a compound of structure (II):

or a pharmaceutically acceptable salt thereof; and

the Compound A is a compound of structure (I):

or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments of any of the above aspects of the invention, thePD-1 antagonist in the combination is an anti-human PD-1 antibody or anantigen binding fragment thereof, and the combination is for use in thetreatment of a cancer in a human. In more preferred embodiments, thecancer is melanoma.

In other embodiments of any of the above aspects of the invention, thecombination comprises each of Compound A and Compound B, the PD-1antagonist is an anti-human PD-1 antibody or an antigen binding fragmentthereof, and the combination is for use in treating a human withadvanced or metastatic melanoma that tests positive for a BRAF V600mutation.

In still other embodiments of any of the above aspects of the invention,the combination does not comprise Compound B, the PD-1 antagonist is ananti-PD-1 antibody or an antigen binding fragment thereof, and thecombination is for use in treating patients with advanced or metastaticmelanoma that tests negative for a BRAF V600 mutation.

In yet other embodiments of any of the above aspects of the invention,the combination does not comprise Compound A, the PD-1 antagonist is ananti-human PD-1 antibody or an antigen binding fragment thereof, and thecombination is for use in treating a human with advanced or metastaticmelanoma that tests positive for a BRAF V600 mutation.

In another aspect of the present invention, there is provided apharmaceutical composition for use in combination with a PD-1 antagonistfor treating a cancer, wherein the pharmaceutical composition comprisesa Compound A and/or a Compound B together with a pharmaceuticallyacceptable diluent or carrier, and wherein the Compound A is a compoundof structure (I):

or a pharmaceutically acceptable salt or solvate thereof; and

the compound B is a compound of structure (II):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the pharmaceutical composition of the inventioncomprises each of Compound A and Compound B, the PD-1 antagonist is ananti-human PD-1 antibody or an antigen binding fragment thereof, and thecancer is advanced melanoma that tests positive for a BRAF V600mutation.

In other embodiments, the pharmaceutical composition of the inventiondoes not comprise Compound B, the PD-1 antagonist is an anti-human PD-1antibody or an antigen binding fragment thereof, and the cancer isadvanced melanoma that tests negative for a BRAF V600 mutation.

In yet other embodiments, the above pharmaceutical composition of theinvention does not comprise Compound A, the PD-1 antagonist is ananti-human PD-1 antibody or an antigen binding fragment thereof, and thecancer is advanced melanoma that tests positive for a BRAF V600mutation.

In yet another aspect of the present invention, there is provided apharmaceutical composition comprising a PD-1 antagonist together with apharmaceutically acceptable diluent or carrier for use in a combinationtherapy for treating a cancer, wherein the combination therapy comprisesthe pharmaceutical composition and one or both of a Compound A and aCompound B, wherein the Compound A is a compound of structure (I):

or a pharmaceutically acceptable salt or solvate thereof; and

wherein the Compound B is a compound of structure (II):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the PD-1 antagonist in a pharmaceutical compositionof the invention is an anti-human PD-1 antibody or an antigen bindingfragment thereof.

In another aspect there is provided the use of a combination comprisingMK-3475 or an antigen binding fragment thereof and one or both of aCompound A and a Compound B in the manufacture of medicaments for use incombination for the treatment of cancer, wherein:

the Compound B is a compound of structure (II)

or a pharmaceutically acceptable salt thereof; and

the Compound A is a compound of structure (I)

or a pharmaceutically acceptable salt or solvate thereof.

In another aspect, there is provided a method of treating cancer in amammal comprising administering to said mammal a combination therapy,wherein the combination therapy comprises a therapeutically effectiveamount of a PD-1 antagonist and one or both of a therapeuticallyeffective amount of a Compound A and a therapeutically effective amountof a Compound B, wherein:

the Compound B is a compound of structure (II):

or a pharmaceutically acceptable salt thereof; and

the Compound A is a compound of structure (I):

or a pharmaceutically acceptable salt or solvate thereof.

In another aspect, there is provided a method of treating cancer in ahuman in need thereof comprising administering to the human acombination therapy, wherein the combination therapy comprises atherapeutically effective amount of a PD-1 antagonist and one or both ofa therapeutically effective amount of a Compound A and a therapeuticallyeffective amount of a Compound B, wherein the PD-1 antagonist is ananti-human PD-1 antibody or an antigen binding fragment thereof, theCompound B isN-{3-[5-(2-Amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamideor a pharmaceutically acceptable salt thereof and the Compound A isN-{3-[3-cyclopropyl-5-(2-fluoro-4-iodo-phenylamino)6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydro-2H-pyrido[4,3-d]pyrimidin-1-yl]phenyl}acetamide,or a pharmaceutically acceptable salt or solvate thereof.

In another aspect, there is provided a method of treating cancer in ahuman in need thereof comprising administering to the human atherapeutically effective amount of a combination therapy comprising aPD-1 antagonist and one or both of a Compound A and a Compound B,wherein the PD-1 antagonist is an anti-human PD-1 antibody or an antigenbinding fragment, Compound B isN-{3-[5-(2-Amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamidemethanesulfonate, and the Compound A isN-{3-[3-cyclopropyl-5-(2-fluoro-4-iodo-phenylamino)6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydro-2H-pyrido[4,3-d]pyrimidin-1-yl]phenyl}acetamidedimethyl sulfoxide solvate.

In a further aspect of the invention is provided a method of treatingcancer in a mammal in need thereof which comprises administering atherapeutically effective amount of a combination of the inventionwherein the combination is administered within a specific period and fora duration of time.

In some embodiments, a treatment method of the invention comprisestreating a human diagnosed with advanced melanoma that tests positivefor a BRAF V600 mutation and the administered combination therapycomprises each of Compound A and Compound B, and an anti-human PD-1antibody.

In some embodiments, a treatment method of the invention comprisestreating a human diagnosed with advanced melanoma that tests negativefor a BRAF V600 mutation, and the administered combination therapycomprises each of Compound A and an anti-human PD-1 monoclonal antibodyas the PD-1 antagonist, but does not comprise Compound B.

In yet other embodiments, a human diagnosed with advanced melanoma thattests positive for a BRAF V600 mutation is treated by administering acombination therapy that comprises each of an anti-human PD-1 monoclonalantibody as the PD-1 antagonist and Compound B, but does not compriseCompound A.

In some embodiments of any of the above aspects of the invention, thecancer tests positive for human PD-L1 expression, the PD-1 antagonist isnivolumab or MK-3475, and in particularly preferred embodiments, thePD-1 antagonist is MK-3475, Compound A is trametinib and Compound B isdabrafenib.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows amino acid sequences of the light chain and heavy chainCDRs for an exemplary anti-PD-1 monoclonal antibody useful in thepresent invention (SEQ ID NOs:1-6).

FIG. 2 shows amino acid sequences of the light chain and heavy chainCDRs for another exemplary anti-PD-1 monoclonal antibody useful in thepresent invention (SEQ ID NOs:7-12).

FIG. 3 shows amino acid sequences of the heavy chain variable region andfull length heavy chain for an exemplary anti-PD-1 monoclonal antibodyuseful in the present invention (SEQ ID NO:13 and SEQ ID NO:14).

FIG. 4 shows amino acid sequences of alternative light chain variableregions for an exemplary anti-PD-1 monoclonal antibody useful in thepresent invention (SEQ ID NOs:15-17).

FIG. 5 shows amino acid sequences of alternative light chains for anexemplary anti-PD-1 monoclonal antibody useful in the present invention(SEQ ID NOs:18-20).

FIG. 6 shows amino acid sequences of the heavy and light chains forMK-3475 (SEQ ID NOs. 21 and 22, respectively).

FIG. 7 shows amino acid sequences of the heavy and light chains fornivolumab (SEQ ID NOs. 23 and 24, respectively).

FIG. 8 compares the anti-tumor effect in tumor-bearing mice ofcombination therapy with a murine anti-mouse PD-1 antibody andtrametinib versus monotherapy with either agent alone, with FIG. 8Ashowing the mean tumor volume at various days during treatment with anisotype antibody+vehicle (Control), the murine anti-PD-1 Ab (Anti-PD-1),trametinib, or concurrent administration of both trametinib andAnti-PD-1, and FIG. 8B showing the tumor volume values for individualmice in each treatment group on the first day of treatment (left graph,Day 0) or after 23 days of treatment (right graph, Day 23).

DETAILED DESCRIPTION OF THE INVENTION I. Abbreviations

Throughout the detailed description and examples of the invention thefollowing abbreviations will be used:

BID One dose twice a dayCDR Complementarity determining regionCHO Chinese hamster ovaryDFS Disease free survivalDTR Dose limiting toxicityFFPE Formalin-fixed, paraffin-embeddedFR Framework region

IgG Immunoglobulin G

IHC Immunohistochemistry or immunohistochemical

MTD Maximum Tolerated Dose NCBI National Center for BiotechnologyInformation NCI National Cancer Institute OR Overall Response OS OverallSurvival PD Progressive Disease PFS Progression Free Survival PR PartialResponse

Q2W One dose every two weeksQ3W One dose every three weeksQD One dose per day

RECIST Response Evaluation Criteria in Solid Tumors SD Stable Disease

VH Immunoglobulin heavy chain variable regionVK Immunoglobulin kappa light chain variable region

II. Definitions

So that the invention may be more readily understood, certain technicaland scientific terms are specifically defined below. Unless specificallydefined elsewhere in this document, all other technical and scientificterms used herein have the meaning commonly understood by one ofordinary skill in the art to which this invention belongs.

As used herein, including the appended claims, the singular forms ofwords such as “a,” “an,” and “the,” include their corresponding pluralreferences unless the context clearly dictates otherwise.

“Administration” and “treatment,” as it applies to an animal, human,experimental subject, cell, tissue, organ, or biological fluid, refersto contact of an exogenous pharmaceutical, therapeutic, diagnosticagent, or composition to the animal, human, subject, cell, tissue,organ, or biological fluid. Treatment of a cell encompasses contact of areagent to the cell, as well as contact of a reagent to a fluid, wherethe fluid is in contact with the cell. “Administration” and “treatment”also means in vitro and ex vivo treatments, e.g., of a cell, by areagent, diagnostic, binding compound, or by another cell. The term“subject” includes any organism, preferably an animal, more preferably amammal (e.g., rat, mouse, dog, cat, rabbit) and most preferably a human.

As used herein, the term “antibody” refers to any form of antibody thatexhibits the desired biological or binding activity. Thus, it is used inthe broadest sense and specifically covers, but is not limited to,monoclonal antibodies (including full length monoclonal antibodies),polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies), humanized, fully human antibodies, chimeric antibodies andcamelized single domain antibodies. “Parental antibodies” are antibodiesobtained by exposure of an immune system to an antigen prior tomodification of the antibodies for an intended use, such as humanizationof an antibody for use as a human therapeutic.

In general, the basic monoclonal antibody structural unit comprises atetramer. Each tetramer includes two identical pairs of polypeptidechains, each pair having one “light” (about 25 kDa) and one “heavy”chain (about 50-70 kDa). The amino-terminal portion of each chainincludes a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The carboxy-terminalportion of the heavy chain may define a constant region primarilyresponsible for effector function. Typically, human light chains areclassified as kappa and lambda light chains. Furthermore, human heavychains are typically classified as mu, delta, gamma, alpha, or epsilon,and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE,respectively. Within light and heavy chains, the variable and constantregions are joined by a “J” region of about 12 or more amino acids, withthe heavy chain also including a “D” region of about 10 more aminoacids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nded. Raven Press, N.Y. (1989).

The variable regions of each light/heavy chain pair form the antibodybinding site. Thus, in general, an intact antibody has two bindingsites. Except in bifunctional or bispecific antibodies, the two bindingsites are, in general, the same.

Typically, the variable domains of both the heavy and light chainscomprise three hypervariable regions, also called complementaritydetermining regions (CDRs), which are located within relativelyconserved framework regions (FR). The CDRs are usually aligned by theframework regions, enabling binding to a specific epitope. In general,from N-terminal to C-terminal, both light and heavy chains variabledomains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignmentof amino acids to each domain is, generally, in accordance with thedefinitions of Sequences of Proteins of Immunological Interest, Kabat,et al.; National Institutes of Health, Bethesda, Md.; 5^(th) ed.; NIHPubl. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat,et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) JMol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.

As used herein, the term “hypervariable region” refers to the amino acidresidues of an antibody that are responsible for antigen-binding. Thehypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (i.e. CDRL1, CDRL2 andCDRL3 in the light chain variable domain and CDRH1, CDRH2 and CDRH3 inthe heavy chain variable domain). See Kabat et al. (1991) Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (defining the CDR regionsof an antibody by sequence); see also Chothia and Lesk (1987) J. Mol.Biol. 196: 901-917 (defining the CDR regions of an antibody bystructure). As used herein, the term “framework” or “FR” residues refersto those variable domain residues other than the hypervariable regionresidues defined herein as CDR residues.

As used herein, unless otherwise indicated, “antibody fragment” or“antigen binding fragment” refers to antigen binding fragments ofantibodies, i.e. antibody fragments that retain the ability to bindspecifically to the antigen bound by the full-length antibody, e.g.fragments that retain one or more CDR regions. Examples of antibodybinding fragments include, but are not limited to, Fab, Fab′, F(ab′)₂,and Fv fragments; diabodies; linear antibodies; single-chain antibodymolecules, e.g., sc-Fv; nanobodies and multispecific antibodies formedfrom antibody fragments.

An antibody that “specifically binds to” a specified target protein isan antibody that exhibits preferential binding to that target ascompared to other proteins, but this specificity does not requireabsolute binding specificity. An antibody is considered “specific” forits intended target if its binding is determinative of the presence ofthe target protein in a sample, e.g. without producing undesired resultssuch as false positives. Antibodies, or binding fragments thereof,useful in the present invention will bind to the target protein with anaffinity that is at least two fold greater, preferably at least tentimes greater, more preferably at least 20-times greater, and mostpreferably at least 100-times greater than the affinity with non-targetproteins. As used herein, an antibody is said to bind specifically to apolypeptide comprising a given amino acid sequence, e.g. the amino acidsequence of a mature human PD-1 or human PD-L1 molecule, if it binds topolypeptides comprising that sequence but does not bind to proteinslacking that sequence. An antibody that specifically binds to aspecified human target protein preferably refers to an antibody thatbinds to that target protein with a K_(D) of 1×10^(−7 M) or less, morepreferably 5×10^(−8 M) or less, more preferably 1×10^(−8 M) or less,more preferably 5×10^(−9 M) or less. A preferred method for determiningthe K_(D) of an antibody is by using surface plasmon resonance.

“Chimeric antibody” refers to an antibody in which a portion of theheavy and/or light chain is identical with or homologous tocorresponding sequences in an antibody derived from a particular species(e.g., human) or belonging to a particular antibody class or subclass,while the remainder of the chain(s) is identical with or homologous tocorresponding sequences in an antibody derived from another species(e.g., mouse) or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity.

“Human antibody” refers to an antibody that comprises humanimmunoglobulin protein sequences only. A human antibody may containmurine carbohydrate chains if produced in a mouse, in a mouse cell, orin a hybridoma derived from a mouse cell. Similarly, “mouse antibody” or“rat antibody” refer to an antibody that comprises only mouse or ratimmunoglobulin sequences, respectively.

“Humanized antibody” refers to forms of antibodies that containsequences from non-human (e.g., murine) antibodies as well as humanantibodies. Such antibodies contain minimal sequence derived fromnon-human immunoglobulin. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the hypervariable loopscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinsequence. The humanized antibody optionally also will comprise at leasta portion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. The prefix “hum”, “hu” or “h” is added to antibodyclone designations when necessary to distinguish humanized antibodiesfrom parental rodent antibodies. The humanized forms of rodentantibodies will generally comprise the same CDR sequences of theparental rodent antibodies, although certain amino acid substitutionsmay be included to increase affinity, increase stability of thehumanized antibody, or for other reasons.

As used herein, the B-Raf inhibitorN-{3-[5-(2-Amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamideor pharmaceutically acceptable salt thereof, is represented by acompound structure (II):

or a pharmaceutically acceptable salt thereof. For convenience, thegroup of possible compound and salts is collectively referred to asCompound B, meaning that reference to Compound B will refer to any ofthe compound or pharmaceutically acceptable salt thereof in thealternative. A particularly preferred B-Raf inhibitor for use in any ofthe aspects of the invention is dabrafenib mesylate, which is known bythe trade name TAFINLAR®. TAFINLAR (dabrafenib) capsules are supplied as50-mg and 75-mg capsules for oral administration, and contain thefollowing inactive ingredients: colloidal silicon dioxide, magnesiumstearate, and microcrystalline cellulose.

The term BRAF V600 mutation means a substitution of glutamate (E) forvaline at position 600 (V600E) and/or a substitution of lysine (K) forvaline at position 600 (V600K). A positive test for a BRAF V600 mutationmeans the detection of the presence of either or both of thesesubstitutions, and a negative test for a BRAF V600 mutation means theabsence of both of these substitutions. A commercially available invitro diagnostics (IVD) kit for detecting BRAF V600E and V600K mutationsis marketed by bioMérieux SA under the tradename the THxID™ BRAF.

“Biotherapeutic agent” means a biological molecule, such as an antibodyor fusion protein, that blocks ligand/receptor signaling in anybiological pathway that supports tumor maintenance and/or growth orsuppresses the anti-tumor immune response.

The terms “cancer”, “cancerous”, or “malignant” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, leukemia, blastoma, and sarcoma. Moreparticular examples of such cancers include squamous cell carcinoma,myeloma, small-cell lung cancer, non-small cell lung cancer, glioma,hodgkin's lymphoma, non-hodgkin's lymphoma, acute myeloid leukemia(AML), multiple myeloma, gastrointestinal (tract) cancer, renal cancer,ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocyticleukemia, colorectal cancer, endometrial cancer, kidney cancer, prostatecancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma,pancreatic cancer, glioblastoma multiforme, cervical cancer, braincancer, stomach cancer, bladder cancer, hepatoma, breast cancer, coloncarcinoma, and head and neck cancer. Particularly preferred cancers thatmay be treated in accordance with the present invention have a positivetest for expression of PD-L1 and either a positive test for a BRAF V600mutation (for embodiments that include a Compound B) or a negative testfor a BRAF V600 mutation (for embodiments that do not include a CompoundB). As used herein, advanced melanoma refers to unresectable Stage III)and metastatic melanoma refers to Stage IV melanoma.

“CDR” or “CDRs” as used herein means complementarity determiningregion(s) in a immunoglobulin variable region, defined using the Kabatnumbering system, unless otherwise indicated.

“Chemotherapeutic agent” is a chemical compound useful in the treatmentof cancer. Classes of chemotherapeutic agents include, but are notlimited to: alkylating agents, antimetabolites, kinase inhibitors,spindle poison plant alkaloids, cytoxic/antitumor antibiotics,topisomerase inhibitors, photosensitizers, anti-estrogens and selectiveestrogen receptor modulators (SERMs), anti-progesterones, estrogenreceptor down-regulators (ERDs), estrogen receptor antagonists,leutinizing hormone-releasing hormone agonists, anti-androgens,aromatase inhibitors, EGFR inhibitors, VEGF inhibitors, anti-senseoligonucleotides that that inhibit expression of genes implicated inabnormal cell proliferation or tumor growth. Chemotherapeutic agentsuseful in the treatment methods of the present invention includecytostatic and/or cytotoxic agents.

“Chothia” as used herein means an antibody numbering system described inAl-Lazikani et al., JMB 273:927-948 (1997).

“Combination of the invention” and “combination therapy of theinvention” refers to a combination of a PD-1 antagonist and one or bothof a BRAF inhibitor, suitably Compound B, and a MEK inhibitor, suitablyCompound A.

“Conservatively modified variants” or “conservative substitution” refersto substitutions of amino acids in a protein with other amino acidshaving similar characteristics (e.g. charge, side-chain size,hydrophobicity/hydrophilicity, backbone conformation and rigidity,etc.), such that the changes can frequently be made without altering thebiological activity or other desired property of the protein, such asantigen affinity and/or specificity. Those of skill in this artrecognize that, in general, single amino acid substitutions innon-essential regions of a polypeptide do not substantially alterbiological activity (see, e.g., Watson et al. (1987) Molecular Biologyof the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4th Ed.)). Inaddition, substitutions of structurally or functionally similar aminoacids are less likely to disrupt biological activity. Exemplaryconservative substitutions are set forth in Table 1 below.

TABLE 1 Exemplary Conservative Amino Acid Substitutions Original residueConservative substitution Ala (A) Gly; Ser Arg (R) Lys; His Asn (N) Gln;His Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn Glu (E) Asp; Gln Gly(G) Ala His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg;His Met (M) Leu; Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser (S) ThrThr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu

“Consists essentially of,” and variations such as “consist essentiallyof” or “consisting essentially of,” as used throughout the specificationand claims, indicate the inclusion of any recited elements or group ofelements, and the optional inclusion of other elements, of similar ordifferent nature than the recited elements, that do not materiallychange the basic or novel properties of the specified dosage regimen,method, or composition. As a non-limiting example, a PD-1 antagonistthat consists essentially of a recited amino acid sequence may alsoinclude one or more amino acids, including substitutions of one or moreamino acid residues, which do not materially affect the properties ofthe binding compound.

“Diagnostic anti-PD-L monoclonal antibody” means a mAb whichspecifically binds to the mature form of the designated PD-L (PD-L1 orPDL2) that is expressed on the surface of certain mammalian cells. Amature PD-L lacks the presecretory leader sequence, also referred to asleader peptide The terms “PD-L” and “mature PD-L” are usedinterchangeably herein, and shall be understood to mean the samemolecule unless otherwise indicated or readily apparent from thecontext.

As used herein, a diagnostic anti-human PD-L1 mAb refers to a monoclonalantibody that specifically binds to mature human PD-L1. A mature humanPD-L1 molecule consists of amino acids 19-290 of the following sequence:

(SEQ ID NO: 25) MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET.

Specific examples of diagnostic anti-human PD-L1 mAbs useful asdiagnostic mAbs for immunohistochemistry (IHC) detection of PD-L1expression in formalin-fixed, paraffin-embedded (FFPE) tumor tissuesections are antibody 20C3 and antibody 22C3, which are described in thecopending international patent application PCT/US13/075932, filed 18Dec. 2013. Another anti-human PD-L1 mAb that has been reported to beuseful for IHC detection of PD-L1 expression in FFPE tissue sections(Chen, B. J. et al., Clin Cancer Res 19: 3462-3473 (2013)) is a rabbitanti-human PD-L1 mAb publicly available from Sino Biological, Inc.(Beijing, P.R. China; Catalog number 10084-R015).

“Framework region” or “FR” as used herein means the immunoglobulinvariable regions excluding the CDR regions.

“Homology” refers to sequence similarity between two polypeptidesequences when they are optimally aligned. When a position in both ofthe two compared sequences is occupied by the same amino acid monomersubunit, e.g., if a position in a light chain CDR of two different Absis occupied by alanine, then the two Abs are homologous at thatposition. The percent of homology is the number of homologous positionsshared by the two sequences divided by the total number of positionscompared ×100. For example, if 8 of 10 of the positions in two sequencesare matched or homologous when the sequences are optimally aligned thenthe two sequences are 80% homologous. Generally, the comparison is madewhen two sequences are aligned to give maximum percent homology. Forexample, the comparison can be performed by a BLAST algorithm whereinthe parameters of the algorithm are selected to give the largest matchbetween the respective sequences over the entire length of therespective reference sequences.

The following references relate to BLAST algorithms often used forsequence analysis: BLAST ALGORITHMS: Altschul, S. F., et al., (1990) J.Mol. Biol. 215:403-410; Gish, W., et al., (1993) Nature Genet.3:266-272; Madden, T. L., et al., (1996) Meth. Enzymol. 266:131-141;Altschul, S. F., et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang,J., et al., (1997) Genome Res. 7:649-656; Wootton, J. C., et al., (1993)Comput. Chem. 17:149-163; Hancock, J. M. et al., (1994) Comput. Appl.Biosci. 10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, M. O., et al., “Amodel of evolutionary change in proteins.” in Atlas of Protein Sequenceand Structure, (1978) vol. 5, suppl. 3. M. O. Dayhoff (ed.), pp.345-352, Natl. Biomed. Res. Found., Washington, DC; Schwartz, R. M., etal., “Matrices for detecting distant relationships.” in Atlas of ProteinSequence and Structure, (1978) vol. 5, suppl. 3.” M. O. Dayhoff (ed.),pp. 353-358, Natl. Biomed. Res. Found., Washington, DC; Altschul, S. F.,(1991) J. Mol. Biol. 219:555-565; States, D. J., et al., (1991) Methods3:66-70; Henikoff, S., et al., (1992) Proc. Natl. Acad. Sci. USA89:10915-10919; Altschul, S. F., et al., (1993) J. Mol. Evol.36:290-300; ALIGNMENT STATISTICS: Karlin, S., et al., (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268; Karlin, S., et al., (1993) Proc. Natl.Acad. Sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob.22:2022-2039; and Altschul, S. F. “Evaluating the statisticalsignificance of multiple distinct local alignments.” in Theoretical andComputational Methods in Genome Research (S. Suhai, ed.), (1997) pp.1-14, Plenum, New York.

“Isolated antibody” and “isolated antibody fragment” refers to thepurification status and in such context means the named molecule issubstantially free of other biological molecules such as nucleic acids,proteins, lipids, carbohydrates, or other material such as cellulardebris and growth media. Generally, the term “isolated” is not intendedto refer to a complete absence of such material or to an absence ofwater, buffers, or salts, unless they are present in amounts thatsubstantially interfere with experimental or therapeutic use of thebinding compound as described herein.

“Kabat” as used herein means an immunoglobulin alignment and numberingsystem pioneered by Elvin A. Kabat ((1991) Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md.).

As used herein, the MEK inhibitorN-{3-[3-cyclopropyl-5-(2-fluoro-4-iodo-phenylamino)6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydro-2H-pyrido[4,3-d]pyrimidin-1-yl]phenyl}acetamide,or a pharmaceutically acceptable salt or solvate thereof, is representedby a compound of structure (I):

or pharmaceutically acceptable salt or solvate thereof. For convenience,the group of possible compound and salts or solvates is collectivelyreferred to as Compound A, meaning that reference to Compound A willrefer to any of the compound or pharmaceutically acceptable salt orsolvate thereof in the alternative. Depending on naming convention, thecompound of formula (I) may also properly be referred to asN-{3-[3-cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-1(2H)-yl]phenyl}acetamide.A particularly preferred MEK inhibitor for use in any of the aspects ofthe invention is trametinib dimethyl sulfoxide, which is known by thetrade name MEKINIST™. MEKINIST (trametinib) tablets are supplied as0.5-mg, 1-mg and 2-mg tablets for oral administration. The core ofMEKINIST tablets contain the inactive ingredients of colloidal silicondioxide, croscarmellose sodium, hypromellse, magnesium stearate(vegetable source) mannitol, microcrystalline cellulose, sodium andlauryl sulfate, and the coating of MEKINIST tablets contain the inactiveingredients of hypromellose, iron oxide red (2-mg tablets), iron oxideyellow (0.5-mg tablets), polyethylene glycol, polysorbate 80 (2-mgtablets) and titanium dioxide.

“Monoclonal antibody” or “mAb” or “Mab”, as used herein, refers to apopulation of substantially homogeneous antibodies, i.e., the antibodymolecules comprising the population are identical in amino acid sequenceexcept for possible naturally occurring mutations that may be present inminor amounts. In contrast, conventional (polyclonal) antibodypreparations typically include a multitude of different antibodieshaving different amino acid sequences in their variable domains,particularly their CDRs, which are often specific for differentepitopes. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al. (1975)Nature 256: 495, or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J.Mol. Biol. 222: 581-597, for example. See also Presta (2005) J. AllergyClin. Immunol. 116:731.

“Patient” or “subject” refers to any single subject for which therapy isdesired or that is participating in a clinical trial, epidemiologicalstudy or used as a control, including humans and mammalian veterinarypatients such as cattle, horses, dogs, and cats.

“PD-1 antagonist” means any chemical compound or biological moleculethat blocks binding of PD-L1 expressed on a cancer cell to PD-1expressed on an immune cell (T cell, B cell or NKT cell) and preferablyalso blocks binding of PD-L2 expressed on a cancer cell to theimmune-cell expressed PD-1. Alternative names or synonyms for PD-1 andits ligands include: PDCD1, PD1, CD279 and SLEB2 for PD-1; PDCD1L1,PDL1, B7H1, B7-4, CD274 and B7-H for PD-L1; and PDCD1L2, PDL2, B7-DC,Btdc and CD273 for PD-L2. In any embodiments of the aspects orembodiments of the present invention in which a human individual is tobe treated, the PD-1 antagonist blocks binding of human PD-L1 to humanPD-1, and preferably blocks binding of both human PD-L1 and PD-L2 tohuman PD-1. Human PD-1 amino acid sequences can be found in NCBI LocusNo.: NP_005009. Human PD-L1 and PD-L2 amino acid sequences can be foundin NCBI Locus No.: NP_054862 and NP_079515, respectively.

PD-1 antagonists useful in the any of the aspects of the presentinvention include a monoclonal antibody (mAb), or antigen bindingfragment thereof, which specifically binds to PD-1 or PD-L1, andpreferably specifically binds to human PD-1 or human PD-L1. The mAb maybe a human antibody, a humanized antibody or a chimeric antibody, andmay include a human constant region. In some embodiments, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG3 and IgG4 constant regions, and in preferred embodiments, the humanconstant region is an IgG1 or IgG4 constant region. In some embodiments,the antigen binding fragment is selected from the group consisting ofFab, Fab′-SH, F(ab′)₂, scFv and Fv fragments.

Examples of mAbs that bind to human PD-1, and useful in the variousaspects and embodiments of the present invention, are described in U.S.Pat. No. 7,488,802, U.S. Pat. No. 7,521,051, U.S. Pat. No. 8,008,449,U.S. Pat. No. 8,354,509, U.S. Pat. No. 8,168,757, WO2004/004771,WO2004/072286, WO2004/056875, and US2011/0271358.

Specific anti-human PD-1 mAbs useful as the PD-1 antagonist in any ofthe aspects and embodiments of the present invention include: MK-3475, ahumanized IgG4 mAb with the structure described in WHO Drug Information,Vol. 27, No. 2, pages 161-162 (2013) and which comprises the heavy andlight chain amino acid sequences shown in FIG. 6; nivolumab, a humanIgG4 mAb with the structure described in WHO Drug Information, Vol. 27,No. 1, pages 68-69 (2013) and which comprises the heavy and light chainamino acid sequences shown in FIG. 7; the humanized antibodies h409A11,h409A16 and h409A17, which are described in WO2008/156712, and AMP-514,which is being developed by Medimmune.

Other PD-1 antagonists useful in the any of the aspects and embodimentsof the present invention include an immunoadhesin that specificallybinds to PD-1, and preferably specifically binds to human PD-1, e.g., afusion protein containing the extracellular or PD-1 binding portion ofPD-L1 or PD-L2 fused to a constant region such as an Fc region of animmunoglobulin molecule. Examples of immunoadhesion molecules thatspecifically bind to PD-1 are described in WO2010/027827 andWO2011/066342. Specific fusion proteins useful as the PD-1 antagonist inthe treatment method, medicaments and uses of the present inventioninclude AMP-224 (also known as B7-DClg), which is a PD-L2-FC fusionprotein and binds to human PD-1.

In some preferred embodiments of any of the aspects of the presentinvention, the PD-1 antagonist is a monoclonal antibody, or antigenbinding fragment thereof, which binds to human PD-1 and comprises: (a)light chain CDRs SEQ ID NOs: 1, 2 and 3 and heavy chain CDRs SEQ ID NOs:4, 5 and 6; or (b) light chain CDRs SEQ ID NOs: 7, 8 and 9 and heavychain CDRs SEQ ID NOs: 10, 11 and 12.

In other preferred embodiments of any of the aspects of the presentinvention, the PD-1 antagonist is a monoclonal antibody, or antigenbinding fragment thereof, which specifically binds to human PD-1 andcomprises (a) a heavy chain variable region comprising SEQ ID NO:13 or avariant thereof, and (b) a light chain variable region comprising anamino acid sequence selected from the group consisting of SEQ ID NO:15or a variant thereof; SEQ ID NO:16 or a variant thereof; and SEQ ID NO:17 or a variant thereof. A variant of a heavy chain variable regionsequence is identical to the reference sequence except having up to 17conservative amino acid substitutions in the framework region (i.e.,outside of the CDRs), and preferably has less than ten, nine, eight,seven, six or five conservative amino acid substitutions in theframework region. A variant of a light chain variable region sequence isidentical to the reference sequence except having up to fiveconservative amino acid substitutions in the framework region (i.e.,outside of the CDRs), and preferably has less than four, three or twoconservative amino acid substitution in the framework region.

In another preferred embodiment of the aspects of the present invention,the PD-1 antagonist is a monoclonal antibody which specifically binds tohuman PD-1 and comprises (a) a heavy chain comprising SEQ ID NO: 14 and(b) a light chain comprising SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.

In yet another preferred embodiment of the aspects of the presentinvention, the PD-1 antagonist is a monoclonal antibody whichspecifically binds to human PD-1 and comprises (a) a heavy chaincomprising SEQ ID NO: 14 and (b) a light chain comprising SEQ ID NO:18.

Table 2 below provides a list of the amino acid sequences of exemplaryanti-PD-1 mAbs for use in the various aspects of the present invention,and the sequences are shown in FIGS. 1-5.

TABLE 2 Exemplary anti-human PD-1 antibodies A. Comprises light andheavy chain CDRs of hPD-1.08A in WO2008/156712 CDRL1 SEQ ID NO: 1 CDRL2SEQ ID NO: 2 CDRL3 SEQ ID NO: 3 CDRH1 SEQ ID NO: 4 CDRH2 SEQ ID NO: 5CDRH3 SEQ ID NO: 6 B. Comprises light and heavy chain CDRs of hPD-1.09Ain WO2008/156712 CDRL1 SEQ ID NO: 7 CDRL2 SEQ ID NO: 8 CDRL3 SEQ ID NO:9 CDRH1 SEQ ID NO: 10 CDRH2 SEQ ID NO: 11 CDRH3 SEQ ID NO: 12 C.Comprises the mature h109A heavy chain variable region and one of themature K09A light chain variable regions in WO2008/156712 Heavy chain VRSEQ ID NO: 13 Light chain VR SEQ ID NO: 15 or SEQ ID NO: 16 or SEQ IDNO: 17 D. Comprises the mature 409 heavy chain and one of the matureK09A light chains in WO2008/156712 Heavy chain SEQ ID NO: 14 Light chainSEQ ID NO: 18 or SEQ ID NO: 19 or SEQ ID NO: 20

“PD-L1” or “PD-L2” expression as used herein means any detectable levelof expression of the designated PD-L protein on the cell surface or ofthe designated PD-L mRNA within a cell or tissue. PD-L proteinexpression may be detected with a diagnostic PD-L antibody in an IHCassay of a tumor tissue section or by flow cytometry. Alternatively,PD-L protein expression by tumor cells may be detected by PET imaging,using a binding agent (e.g., antibody fragment, affibody and the like)that specifically binds to the desired PD-L target, e.g., PD-L1 orPD-L2. Techniques for detecting and measuring PD-L mRNA expressioninclude RT-PCR and realtime quantitative RT-PCR.

Several approaches have been described for quantifying PD-L1 proteinexpression in IHC assays of tumor tissue sections. See, e.g., Thompson,R. H., et al., PNAS 101 (49); 17174-17179 (2004); Thompson, R. H. etal., Cancer Res. 66:3381-3385 (2006); Gadiot, J., et al., Cancer117:2192-2201 (2011); Taube, J. M. et al., Sci Transl Med 4, 127ra37(2012); and Toplian, S. L. et al., New Eng. J Med. 366 (26): 2443-2454(2012).

One approach employs a simple binary end-point of positive or negativefor PD-L1 expression, with a positive result defined in terms of thepercentage of tumor cells that exhibit histologic evidence ofcell-surface membrane staining. A tumor tissue section is counted aspositive for PD-L1 expression is at least 1%, and preferably 5% of totaltumor cells.

In another approach, PD-L1 expression in the tumor tissue section isquantified in the tumor cells as well as in infiltrating immune cells,which predominantly comprise lymphocytes. The percentage of tumor cellsand infiltrating immune cells that exhibit membrane staining areseparately quantified as <5%, 5 to 9%, and then in 10% increments up to100%. For tumor cells, PD-L1 expression is counted as negative if thescore is <5% score and positive if the score is 5%. PD-L1 expression inthe immune infiltrate is reported as a semi-quantitative measurementcalled the adjusted inflammation score (AIS), which is determined bymultiplying the percent of membrane staining cells by the intensity ofthe infiltrate, which is graded as none (0), mild (score of 1, rarelymphocytes), moderate (score of 2, focal infiltration of tumor bylymphohistiocytic aggregates), or severe (score of 3, diffuseinfiltration). A tumor tissue section is counted as positive for PD-L1expression by immune infiltrates if the AIS is 5.

The level of PD-L mRNA expression may be compared to the mRNA expressionlevels of one or more reference genes that are frequently used inquantitative RT-PCR, such as ubiquitin C.

In some embodiments, a level of PD-L1 expression (protein and/or mRNA)by malignant cells and/or by infiltrating immune cells within a tumor isdetermined to be “overexpressed” or “elevated” based on comparison withthe level of PD-L1 expression (protein and/or mRNA) by an appropriatecontrol. For example, a control PD-L1 protein or mRNA expression levelmay be the level quantified in nonmalignant cells of the same type or ina section from a matched normal tissue. In some preferred embodiments,PD-L1 expression in a tumor sample is determined to be elevated if PD-L1protein (and/or PD-L1 mRNA) in the sample is at least 10%, 20%, or 30%greater than in the control.

“RECIST 1.1 Response Criteria” as used herein means the definitions setforth in Eisenhauer et al., E. A. et al., Eur. J Cancer 45:228-247(2009) for target lesions or nontarget lesions, as appropriate based onthe context in which response is being measured.

“Sustained response” means a sustained therapeutic effect aftercessation of treatment with a therapeutic agent, or a combinationtherapy described herein. In some embodiments, the sustained responsehas a duration that is at least the same as the treatment duration, orat least 1.5, 2.0, 2.5 or 3 times longer than the treatment duration.

“Tissue Section” refers to a single part or piece of a tissue sample,e.g., a thin slice of tissue cut from a sample of a normal tissue or ofa tumor.

“Treat” or “treating” a cancer as used herein means to administer acombination of the invention to a subject having a cancer, or diagnosedwith a cancer, to achieve at least one positive therapeutic effect, suchas for example, reduced number of cancer cells, reduced tumor size,reduced rate of cancer cell infiltration into peripheral organs, orreduced rate of tumor metastasis or tumor growth. Positive therapeuticeffects in cancer can be measured in a number of ways (See, W. A. Weber,J. Nucl. Med. 50:1S-10S (2009)). For example, with respect to tumorgrowth inhibition, according to NCI standards, a T/C≦42% is the minimumlevel of anti-tumor activity. A T/C<10% is considered a high anti-tumoractivity level, with T/C (%)=Median tumor volume of the treated/Mediantumor volume of the control×100. In some embodiments, the treatmentachieved by a combination of the invention is any of PR, CR, OR, PFS,DFS and OS. PFS, also referred to as “Time to Tumor Progression”indicates the length of time during and after treatment that the cancerdoes not grow, and includes the amount of time patients have experienceda CR or PR, as well as the amount of time patients have experienced SD.DFS refers to the length of time during and after treatment that thepatient remains free of disease. OS refers to a prolongation in lifeexpectancy as compared to naive or untreated individuals or patients. Insome preferred embodiments, response to a combination of the inventionis any of PR, CR, PFS, DFS, OR or OS that is assessed using RECIST 1.1response criteria. The treatment regimen for a combination of theinvention that is effective to treat a cancer patient may vary accordingto factors such as the disease state, age, and weight of the patient,and the ability of the therapy to elicit an anti-cancer response in thesubject. While an embodiment of any of the aspects of the invention maynot be effective in achieving a positive therapeutic effect in everysubject, it should do so in a statistically significant number ofsubjects as determined by any statistical test known in the art such asthe Student's t-test, the chi²-test, the U-test according to Mann andWhitney, the Kruskal-Wallis test (H-test), Jonckheere-Terpstra-test andthe Wilcoxon-test.

The terms “treatment regimen”, “dosing protocol” and dosing regimen areused interchangeably to refer to the dose and timing of administrationof each therapeutic agent in a combination of the invention.

“Tumor” or “neoplasm” as applied to a subject diagnosed with, orsuspected of having, a cancer refers to a malignant or potentiallymalignant neoplasm or tissue mass of any size, and includes primarytumors and secondary neoplasms. A solid tumor is an abnormal growth ormass of tissue that usually does not contain cysts or liquid areas.Different types of solid tumors are named for the type of cells thatform them. Examples of solid tumors are sarcomas, carcinomas, andlymphomas. Leukemias (cancers of the blood) generally do not form solidtumors (National Cancer Institute, Dictionary of Cancer Terms).

“Tumor burden” also referred to as “tumor load”, refers to the totalamount of tumor material distributed throughout the body. Tumor burdenrefers to the total number of cancer cells or the total size oftumor(s), throughout the body, including lymph nodes and bone narrow.Tumor burden can be determined by a variety of methods known in the art,such as, e.g. by measuring the dimensions of tumor(s) upon removal fromthe subject, e.g., using calipers, or while in the body using imagingtechniques, e.g., ultrasound, bone scan, computed tomography (CT) ormagnetic resonance imaging (MRI) scans.

The term “tumor size” refers to the total size of the tumor which can bemeasured as the length and width of a tumor. Tumor size may bedetermined by a variety of methods known in the art, such as, e.g. bymeasuring the dimensions of tumor(s) upon removal from the subject,e.g., using calipers, or while in the body using imaging techniques,e.g., bone scan, ultrasound, CT or MRI scans.

“Variable regions” or “V region” as used herein means the segment of IgGchains which is variable in sequence between different antibodies. Itextends to Kabat residue 109 in the light chain and 113 in the heavychain.

III. Combinations, Compositions, Uses and Treatment Methods

The administration of a therapeutically effective amount of thecombinations of the invention are advantageous over the individualcomponent compounds in that the combinations provide one or more of thefollowing improved properties when compared to the individualadministration of a therapeutically effective amount of a componentcompound: i) a greater anticancer effect than the most active singleagent, ii) synergistic or highly synergistic anticancer activity, iii) adosing protocol that provides enhanced anticancer activity with reducedside effect profile, iv) a reduction in the toxic effect profile, v) anincrease in the therapeutic window, or vi) an increase in thebioavailability of one or both of the component compounds.

Compounds A and/or B may contain one or more chiral atoms, or mayotherwise be capable of existing as enantiomers. Accordingly, thecompounds of this invention include mixtures of enantiomers as well aspurified enantiomers or enantiomerically enriched mixtures. Also, it isunderstood that all tautomers and mixtures of tautomers are includedwithin the scope of Compound A and Compound B.

Also, it is understood that compounds A and B may be presented,separately or both, as solvates. As used herein, the term “solvate”refers to a complex of variable stoichiometry formed by a solute (inthis invention, compounds of formula (I) or (II) or a salt thereof and asolvent. Such solvents for the purpose of the invention may notinterfere with the biological activity of the solute. Examples ofsuitable solvents include, but are not limited to, water, methanol,dimethyl sulfoxide, ethanol and acetic acid. In one embodiment, thesolvent used is a pharmaceutically acceptable solvent. In anotherembodiment, the solvent used is water.

Compounds A and B may have the ability to crystallize in more than oneform, a characteristic, which is known polymorphism, and it isunderstood that such polymorphic forms (“polymorphs”) are within thescope of Compounds A and B. Polymorphism generally can occur as aresponse to changes in temperature or pressure or both and can alsoresult from variations in the crystallization process. Polymorphs can bedistinguished by various physical characteristics known in the art suchas x-ray diffraction patterns, solubility, and melting point.

Compound A is disclosed and claimed, along with pharmaceuticallyacceptable salts and solvates thereof, as being useful as an inhibitorof MEK activity, particularly in treatment of cancer, in InternationalApplication No. PCT/JP2005/011082, having an International filing dateof Jun. 10, 2005; International Publication Number WO 2005/121142 and anInternational Publication date of Dec. 22, 2005, the entire disclosureof which is hereby incorporated by reference, Compound A is the compoundof Example 4-1. Compound A can be prepared as described in InternationalApplication No. PCT/JP2005/011082. Compound A can be prepared asdescribed in United States Patent Publication No. US 2006/0014768,published Jan. 19, 2006, the entire disclosure of which is herebyincorporated by reference.

Suitably, Compound A is in the form of a dimethyl sulfoxide solvate.Suitably, Compound A is in the form of a sodium salt. Suitably, CompoundA is in the form of a solvate selected from: hydrate, acetic acid,ethanol, nitromethane, chlorobenzene, 1-pentanci, isopropyl alcohol,ethylene glycol and 3-methyl-1-butanol. These solvates and salt formscan be prepared by one of skill in the art from the description inInternational Application No. PCT/JP2005/011082 or United States PatentPublication No. US 2006/0014768.

Compound B is disclosed and claimed, along with pharmaceuticallyacceptable salts thereof, as being useful as an inhibitor of BRafactivity, particularly in the treatment of cancer, in PCT patentapplication PCT/US09/42682. Compound B is embodied by Examples 58athrough 58e of the application. The PCT application was published on 12Nov. 2009 as publication WO2009/137391, and is hereby incorporated byreference.

More particularly, Compound B may be prepared according to the methodsbelow:

Method 1: Compound B (first crystalform)—N-{3-[5-(2-Amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide

A suspension ofN-{3-[5-(2-chloro-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide(196 mg, 0.364 mmol) and ammonia in methanol 7M (8 ml, 56.0 mmol) washeated in a sealed tube to 90° C. for 24 h. The reaction was dilutedwith DCM and added silica gel and concentrated. The crude product waschromatographed on silica gel eluting with 100% DCM to 1:1 [DCM:(9:1EtOAc:MeOH)]. The clean fractions were concentrated to yield the crudeproduct. The crude product was repurified by reverse phase HPLC (agradient of acetonitrile:water with 0.1% TFA in both). The combinedclean fractions were concentrated then partitioned between DCM andsaturated NaHCO₃. The DCM layer was separated and dried over Na₂SO₄. Thetitle compound,N-{3-[5-(2-amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamidewas obtained (94 mg, 47% yield). ¹H NMR (400 MHz, DMSO-d6) δ ppm 10.83(s, 1H), 7.93 (d, J=5.2 Hz, 1H), 7.55-7.70 (m, 1H), 7.35-7.43 (m, 1H),7.31 (t, J=6.3 Hz, 1H), 7.14-7.27 (m, 3H), 6.70 (s, 2H), 5.79 (d, J=5.13Hz, 1H), 1.35 (s, 9H). MS (ESI): 519.9 [M+H]⁺.

Method 2: Compound B (alternative crystalform)—N-{3-[5-(2-Amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide19.6 mg ofN-{3-[5-(2-Amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide(may be prepared in accordance with example 58a) was combined with 500 Lof ethyl acetate in a 2-mL vial at room temperature. The slurry wastemperature-cycled between 0-40° C. for 48 hrs. The resulting slurry wasallowed to cool to room temperature and the solids were collected byvacuum filtration. The solids were analyzed by Raman, PXRD, DSC/TGAanalyses, which indicated a crystal form different from the crystal formresulting from Example 58a, above.

Method 3: Compound B (alternative crystal form, largebatch)—N-{3-[5-(2-amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide

Step A: methyl 3-{[(2,6-difluorophenyl)sulfonyl]amino}-2-fluorobenzoate

Methyl 3-amino-2-fluorobenzoate (50 g, 1 eq) was charged to reactorfollowed by dichloromethane (250 mL, 5 vol). The contents were stirredand cooled to ˜15° C. and pyridine (26.2 mL, 1.1 eq) was added. Afteraddition of the pyridine, the reactor contents were adjusted to ˜15° C.and the addition of 2,6-diflurorobenzenesulfonyl chloride (39.7 mL, 1.0eq) was started via addition funnel. The temperature during addition waskept <25° C. After complete addition, the reactor contents were warmedto 20-25° C. and held overnight. Ethyl acetate (150 mL) was added anddichloromethane was removed by distillation. Once distillation wascomplete, the reaction mixture was then diluted once more with ethylacetate (5 vol) and concentrated. The reaction mixture was diluted withethyl acetate (10 vol) and water (4 vol) and the contents heated to50-55° C. with stirring until all solids dissolve. The layers weresettled and separated. The organic layer was diluted with water (4 vol)and the contents heated to 50-55° for 20-30 min. The layers were settledand then separated and the ethyl acetate layer was evaporated underreduced pressure to ˜3 volumes. Ethyl Acetate (5 vol.) was added andagain evaporated under reduced pressure to ˜3 volumes. Cyclohexane (9vol) was then added to the reactor and the contents were heated toreflux for 30 min then cooled to 0° C. The solids were filtered andrinsed with cyclohexane (2×100 mL). The solids were air dried overnightto obtain methyl3-{[(2,6-difluorophenyl)sulfonyl]amino}-2-fluorobenzoate (94.1 g, 91%).

Step B:N-{3-[(2-chloro-4-pyrimidinyl)acetyl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide

Methyl 3-{[(2,6-difluorophenyl)sulfonyl]amino}-2-fluorobenzoate (490 g,1 equiv.), prepared generally in accordance with Step A, above, wasdissolved in THF (2.45 L, 5 vols) and stirred and cooled to 0-3° C. 1Mlithium bis(trimethylsilyl)amide in THF (5.25 L, 3.7 equiv.) solutionwas charged to the reaction mixture followed addition of2-chloro-4-methylpyrimidine (238 g, 1.3 equiv.) in THF (2.45 L, 5 vols).The reaction was then stirred for 1 hr. The reaction was quenched with4.5M HCl (3.92 L, 8 vols). The aqueous layer (bottom layer) was removedand discarded. The organic layer was concentrated under reduced pressureto ˜2 L. IPAC (isopropyl acetate) (2.45 L) was added to the reactionmixture which was then concentrated to ˜2 L. IPAC (0.5 L) and MTBE (2.45L) was added and stirred overnight under N₂. The solids were filtered.The solids and mother filtrate added back together and stirred forseveral hours. The solids were filtered and washed with MTBE (˜5 vol).The solids were placed in vacuum oven at 50° C. overnight. The solidswere dried in vacuum oven at 30° C. over weekend to obtainN-{3-[(2-chloro-4-pyrimidinyl)acetyl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide(479 g, 72%).

Step C:N-{3-[5-(2-chloro-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide

To a reactor vessel was chargedN-{3-[(2-chloro-4-pyrimidinyl)acetyl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide(30 g, 1 eq) followed by dichloromethane (300 mL). The reaction slurrywas cooled to ˜10° C. and N-bromosuccinimide (“NBS”) (12.09 g, 1 eq) wasadded in 3 approximately equal portions, stirring for 10-15 minutesbetween each addition. After the final addition of NBS, the reactionmixture was warmed to ˜20° C. and stirred for 45 min. Water (5 vol) wasthen added to the reaction vessel and the mixture was stirred and thenthe layers separated. Water (5 vol) was again added to thedichloromethane layer and the mixture was stirred and the layersseparated. The dichloromethane layers were concentrated to ˜120 mL.Ethyl acetate (7 vol) was added to the reaction mixture and concentratedto ˜120 mL. Dimethylacetamide (270 mL) was then added to the reactionmixture and cooled to ˜10° C. 2,2-Dimethylpropanethioamide (1.3 g, 0.5eq) in 2 equal portions was added to the reactor contents with stirringfor ˜5 minutes between additions. The reaction was warmed to 20-25° C.After 45 min, the vessel contents were heated to 75° C. and held for1.75 hours. The reaction mixture was then cooled to 5° C. and water (270ml) was slowly charged keeping the temperature below 30° C. Ethylacetate (4 vol) was then charged and the mixture was stirred and layersseparated. Ethyl acetate (7 vol) was again charged to the aqueous layerand the contents were stirred and separated. Ethyl acetate (7 vol) wascharged again to the aqueous layer and the contents were stirred andseparated. The organic layers were combined and washed with water (4vol) 4 times and stirred overnight at 20-25° C. The organic layers werethen concentrated under heat and vacuum to 120 mL. The vessel contentswere then heated to 50° C. and heptanes (120 mL) were added slowly.After addition of heptanes, the vessel contents were heated to refluxthen cooled to 0° C. and held for ˜2 hrs. The solids were filtered andrinsed with heptanes (2×2 vol). The solid product was then dried undervacuum at 30° C. to obtainN-{3-[5-(2-chloro-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide(28.8 g, 80%).

Step D:N-{3-[5-(2-amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide

In 1 gal pressure reactor, a mixture ofN-{3-[5-(2-chloro-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide(120 g) prepared in accordance with Step C, above, and ammoniumhydroxide (28-30%, 2.4 L, 20 vol) was heated in the sealed pressurereactor to 98-103° C. and stirred at this temperature for 2 hours. Thereaction was cooled slowly to room temperature (20° C.) and stirredovernight. The solids were filtered and washed with minimum amount ofthe mother liquor and dried under vacuum. The solids were added to amixture of EtOAc (15 vol)/water (2 vol) and heated to completedissolution at 60-70° C. and the aqueous layer was removed anddiscarded. The EtOAC layer was charged with water (1 vol) andneutralized with aq. HCl to ˜pH 5.4-5.5. and added water (1 vol). Theaqueous layer was removed and discarded at 60-70° C. The organic layerwas washed with water (1 vol) at 60-70° C. and the aqueous layer wasremoved and discarded. The organic layer was filtered at 60° C. andconcentrated to 3 volumes. EtOAc (6 vol) was charged into the mixtureand heated and stirred at 72° C. for 10 min, then cooled to 20° C. andstirred overnight. EtOAc was removed via vacuum distillation toconcentrate the reaction mixture to ˜3 volumes. The reaction mixture wasmaintained at ˜65-70° C. for ˜30 mins. Product crystals having the samecrystal form as those prepared in Example 58b (and preparable by theprocedure of Example 58b), above, in heptanes slurry were charged.Heptane (9 vol) was slowly added at 65-70° C. The slurry was stirred at65-70° C. for 2-3 hours and then cooled slowly to 0-5° C. The productwas filtered, washed with EtOAc/heptane (3/1 v/v, 4 vol) and dried at45° C. under vacuum to obtainN-{3-[5-(2-amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide(102.3 g, 88%).

Method 4: Compound B (mesylatesalt)—N-{3-[5-(2-amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamidemethanesulfonate

To a solution ofN-{3-[5-(2-amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide(204 mg, 0.393 mmol) in isopropanol (2 mL), methanesulfonic acid (0.131mL, 0.393 mmol) was added and the solution was allowed to stir at roomtemperature for 3 hours. A white precipitate formed and the slurry wasfiltered and rinsed with diethyl ether to give the title product as awhite crystalline solid (210 mg, 83% yield). ¹H NMR (400 MHz, DMSO-d6) δppm 10.85 (s, 1H) 7.92-8.05 (m, 1H) 7.56-7.72 (m, 1H) 6.91-7.50 (m, 7H)5.83-5.98 (m, 1H) 2.18-2.32 (m, 3H) 1.36 (s, 9H). MS (ESI): 520.0[M+H]⁺.

Method 5: Compound B (alternative mesylate saltembodiment)—N-{3-[5-(2-amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamidemethanesulfonate

N-{3-[5-(2-amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide(as may be prepared according to example 58a) (2.37 g, 4.56 mmol) wascombined with pre-filtered acetonitrile (5.25 vol, 12.4 mL). Apre-filtered solution of mesic acid (1.1 eq., 5.02 mmol, 0.48 g) in H₂O(0.75 eq., 1.78 mL) was added at 20° C. The temperature of the resultingmixture was raised to 50-60° C. while maintaining a low agitation speed.Once the mixture temperature reached to 50-60° C., a seed slurry ofN-{3-[5-(2-amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamidemethanesulfonate (1.0% w/w slurried in 0.2 vol of pre-filteredacetonitrile) was added, and the mixture was aged while agitating at aspeed fast enough to keep solids from settling at 50-60° C. for 2 hr.The mixture was then cooled to 0-5° C. at 0.25° C./min and held at 0-5°C. for at 6 hr. The mixture was filtered and the wet cake was washedtwice with pre-filtered acetonitrile. The first wash consisted of 14.2ml (6 vol) pre-filtered acetonitrile and the second wash consisted of9.5 ml (4 vol) pre-filtered acetonitrile. The wet solid was dried at 50°C. under vacuum, yielding 2.39 g (85.1% yield) of product.

Typically, the salts of the present invention are pharmaceuticallyacceptable salts. Salts encompassed within the term “pharmaceuticallyacceptable salts” refer to non-toxic salts of the compounds of thisinvention. Salts of the compounds of the present invention may compriseacid addition salts derived from a nitrogen on a substituent in acompound of the present invention. Representative salts include thefollowing salts: acetate, benzenesulfonate, benzoate, bicarbonate,bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate,carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate,edisylate, estolate, esylate, fumarate, gluceptate, gluconate,glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate,lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate,methylbromide, methylnitrate, methylsulfate, monopotassium maleate,mucate, napsylate, nitrate, N-methylglucamine, oxalate, pamoate(embonate), palmitate, pantothenate, phosphate/diphosphate,polygalacturonate, potassium, salicylate, sodium, stearate, subacetate,succinate, tannate, tartrate, teoclate, tosylate, triethiodide,trimethylammonium and valerate. Other salts, which are notpharmaceutically acceptable, may be useful in the preparation ofcompounds of this invention and these form a further aspect of theinvention. Salts may be readily prepared by a person skilled in the art.

While it is possible that, for use in therapy, compounds A and B may beadministered as the raw chemical, it is possible to present the activeingredient as a pharmaceutical composition. Accordingly, the inventionfurther provides pharmaceutical compositions, which include a Compound Aand/or a Compound B, and one or more pharmaceutically acceptablecarriers, diluents, or excipients. The Compounds A and B are asdescribed above. The carrier(s), diluent(s) or excipient(s) must beacceptable in the sense of being compatible with the other ingredientsof the formulation, capable of pharmaceutical formulation, and notdeleterious to the recipient thereof. In accordance with another aspectof the invention there is also provided a process for the preparation ofa pharmaceutical composition including admixing a Compound A and/orCompound B, with one or more pharmaceutically acceptable carriers,diluents or excipients. Such elements of the pharmaceutical compositionsutilized may be presented in separate pharmaceutical combinations orformulated together in one pharmaceutical composition. Accordingly, theinvention further provides a combination of pharmaceutical compositionsone of which includes Compound A and one or more pharmaceuticallyacceptable carriers, diluents, or excipients and a pharmaceuticalcomposition containing Compound B and one or more pharmaceuticallyacceptable carriers, diluents, or excipients.

A pharmaceutical composition comprising Compound A and/or Compound B maybe used in combination with a pharmaceutical composition comprising thePD-1 antagonist. Similarly, each of the agents in a combination of theinvention may be formulated as separate pharmaceutical compositions foruse in combination with each other.

Pharmaceutical compositions may be presented in unit dose formscontaining a predetermined amount of active ingredient per unit dose. Asis known to those skilled in the art, the amount of active ingredientper dose will depend on the condition being treated, the route ofadministration and the age, weight and condition of the patient.Preferred unit dosage compositions are those containing a daily dose orsub-dose, or an appropriate fraction thereof, of an active ingredient.Furthermore, such pharmaceutical compositions may be prepared by any ofthe methods well known in the pharmacy art.

Compounds A and B may be administered by any appropriate route. Suitableroutes include oral, rectal, nasal, topical (including buccal andsublingual), vaginal, and parenteral (including subcutaneous,intramuscular, intraveneous, intradermal, intrathecal, and epidural). Itwill be appreciated that the preferred route may vary with, for example,the condition of the recipient of the combination and the cancer to betreated. It will also be appreciated that each of the agentsadministered may be administered by the same or different routes andthat the Compounds A and B may be compounded together or in separatepharmaceutical compositions.

Any biotherapeutic agent in a combination of the invention, such as ananti-PD-1 antibody, may be administered parenterally, including byintravenous (IV) infusion and subcutaneous injection.

Pharmaceutical compositions adapted for oral administration may bepresented as discrete units such as capsules or tablets; powders orgranules; solutions or suspensions in aqueous or non-aqueous liquids;edible foams or whips; or oil-in-water liquid emulsions or water-in-oilliquid emulsions.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Powders are prepared by comminuting thecompound to a suitable fine size and mixing with a similarly comminutedpharmaceutical carrier such as an edible carbohydrate, as, for example,starch or mannitol. Flavoring, preservative, dispersing and coloringagent can also be present.

Capsules are made by preparing a powder mixture as described above, andfilling formed gelatin sheaths. Glidants and lubricants such ascolloidal silica, talc, magnesium stearate, calcium stearate or solidpolyethylene glycol can be added to the powder mixture before thefilling operation. A disintegrating or solubilizing agent such asagar-agar, calcium carbonate or sodium carbonate can also be added toimprove the availability of the medicament when the capsule is ingested.

Moreover, when desired or necessary, suitable binders, lubricants,disintegrating agents and coloring agents can also to granulating, thepowder mixture can be run through the tablet machine and the result isimperfectly formed slugs broken into granules. The granules can belubricated be incorporated into the mixture. Suitable binders includestarch, gelatin, natural sugars such as glucose or beta-lactose, cornsweeteners, natural and synthetic gums such as acacia, tragacanth orsodium alginate, carboxymethylcellulose, polyethylene glycol, waxes andthe like. Lubricants used in these dosage forms include sodium oleate,sodium stearate, magnesium stearate, sodium benzoate, sodium acetate,sodium chloride and the like. Disintegrators include, withoutlimitation, starch, methyl cellulose, agar, bentonite, xanthan gum andthe like. Tablets are formulated, for example, by preparing a powdermixture, granulating or slugging, adding a lubricant and disintegrantand pressing into tablets. A powder mixture is prepared by mixing thecompound, suitably comminuted, with a diluent or base as describedabove, and optionally, with a binder such as carboxymethylcellulose, analiginate, gelatin, or polyvinyl pyrrolidone, a solution retardant suchas paraffin, a resorption accelerator such as a quaternary salt and/oran absorption agent such as bentonite, kaolin or dicalcium phosphate.The powder mixture can be granulated by wetting with a binder such assyrup, starch paste, acadia mucilage or solutions of cellulosic orpolymeric materials and forcing through a screen. As an alternative toprevent sticking to the tablet forming dies by means of the addition ofstearic acid, a stearate salt, talc or mineral oil. The lubricatedmixture is then compressed into tablets. The compounds of the presentinvention can also be combined with free flowing inert carrier andcompressed into tablets directly without going through the granulatingor slugging steps. A clear or opaque protective coating consisting of asealing coat of shellac, a coating of sugar or polymeric material and apolish coating of wax can be provided. Dyestuffs can be added to thesecoatings to distinguish different unit dosages.

Oral fluids such as solution, syrups and elixirs can be prepared indosage unit form so that a given quantity contains a predeterminedamount of the compound. Syrups can be prepared by dissolving thecompound in a suitably flavored aqueous solution, while elixirs areprepared through the use of a non-toxic alcoholic vehicle. Suspensionscan be formulated by dispersing the compound in a non-toxic vehicle.Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols andpolyoxy ethylene sorbitol ethers, preservatives, flavor additive such aspeppermint oil or natural sweeteners or saccharin or other artificialsweeteners, and the like can also be added.

Where appropriate, compositions for oral administration can bemicroencapsulated. The composition can also be prepared to prolong orsustain the release as for example by coating or embedding particulatematerial in polymers, wax or the like.

The agents for use according to the present invention can also beadministered in the form of liposome delivery systems, such as smallunilamellar vesicles, large unilamellar vesicles and multilamellarvesicles. Liposomes can be formed from a variety of phospholipids, suchas cholesterol, stearylamine or phosphatidylcholines.

Agents for use according to the present invention may also be deliveredby the use of monoclonal antibodies as individual carriers to which thecompound molecules are coupled. The compounds may also be coupled withsoluble polymers as targetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxypropylmethacrylamide-phenol,polyhydroxyethylaspartamidephenol, or polyethyleneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds may becoupled to a class of biodegradable polymers useful in achievingcontrolled release of a drug, for example, polylactic acid, polepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacrylates and cross-linked or amphipathicblock copolymers of hydrogels.

Pharmaceutical compositions adapted for transdermal administration maybe presented as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time. Forexample, the active ingredient may be delivered from the patch byiontophoresis as generally described in Pharmaceutical Research, 3(6),318 (1986).

Pharmaceutical compositions adapted for topical administration may beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, sprays, aerosols or oils.

For treatments of the eye or other external tissues, for example mouthand skin, the compositions are preferably applied as a topical ointmentor cream. When formulated in an ointment, the active ingredient may beemployed with either a paraffinic or a water-miscible ointment base.Alternatively, the active ingredient may be formulated in a cream withan oil-in-water cream base or a water-in-oil base.

Pharmaceutical compositions adapted for topical administrations to theeye include eye drops wherein the active ingredient is dissolved orsuspended in a suitable carrier, especially an aqueous solvent.

Pharmaceutical compositions adapted for topical administration in themouth include lozenges, pastilles and mouth washes.

Pharmaceutical compositions adapted for rectal administration may bepresented as suppositories or as enemas.

Pharmaceutical compositions adapted for nasal administration wherein thecarrier is a solid include a coarse powder having a particle size forexample in the range 20 to 500 microns which is administered in themanner in which snuff is taken, i.e. by rapid inhalation through thenasal passage from a container of the powder held close up to the nose.Suitable compositions wherein the carrier is a liquid, foradministration as a nasal spray or as nasal drops, include aqueous oroil solutions of the active ingredient.

Pharmaceutical compositions adapted for administration by inhalationinclude fine particle dusts or mists that may be generated by means ofvarious types of metered dose pressurised aerosols, nebulizers orinsufflators.

Pharmaceutical compositions adapted for vaginal administration may bepresented as pessaries, tampons, creams, gels, pastes, foams or spraycompositions.

Pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe formulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The compositions may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets.

When the PD-1 antagonist is a biotherapeutic agent, e.g., a monoclonalantibody that binds to PD-1 or PD-L1, the antagonist may be produced inCHO cells using conventional cell culture and then isolated usingconventional recovery/purification technologies. A pharmaceuticalcomposition comprising an anti-PD-1 antibody as the PD-1 antagonist maybe provided as a liquid formulation or prepared by reconstituting alyophilized powder with sterile water for injection prior to use. WO2012/135408 describes the preparation of liquid and lyophilizedmedicaments comprising MK-3475 that are suitable for use in the presentinvention. In some preferred embodiments, a medicament comprisingMK-3475 is provided in a glass vial which contains about 50 mg ofMK-3475.

It should be understood that in addition to the ingredients particularlymentioned above, the compositions may include other agents conventionalin the art having regard to the type of formulation in question, forexample those suitable for oral administration may include flavoringagents.

Compounds A and B may be employed in combination in accordance with theinvention by administration simultaneously in a unitary pharmaceuticalcomposition including both compounds. Alternatively, the combination maybe administered separately in separate pharmaceutical compositions, eachincluding one of the compounds A and B in a sequential manner wherein,for example, Compound A or Compound B is administered first and theother second. Such sequential administration may be close in time (eg.simultaneously) or remote in time. Furthermore, it does not matter ifthe compounds are administered in the same dosage form, e.g. onecompound may be administered topically and the other compound may beadministered orally. Suitably, both compounds are administered orally.

Thus in one embodiment, one or more doses of Compound A are administeredsimultaneously or separately with one or more doses of Compound B andone or more doses of a PD-1 antagonist.

In one embodiment, multiple doses of Compound A are administeredsimultaneously or separately with multiple doses of Compound B andmultiple doses of MK-3475 or an antigen binding fragment thereof.

In one embodiment, multiple doses of Compound A are administeredsimultaneously or separately with one dose of Compound B and one dose ofnivolumab or an antigen binding fragment thereof or MK-3475 or anantigen binding fragment thereof.

In all the above embodiments, Compound A may be administered first orCompound B may be administered first or the PD-1 antagonist may beadministered first.

The combinations may be presented as a combination kit. By the term“combination kit” “or kit of parts” as used herein is meant thepharmaceutical composition or compositions that are used to administerCompound B, and/or Compound A, and the PD-1 antagonist, e.g., ananti-PD-1 antibody or antigen binding fragment thereof, according to adosage protocol of the invention. When compounds A, B, and an anti-PD-1antibody or antigen binding fragment thereof are administeredsimultaneously, the combination kit can contain Compound A and CompoundB in a single pharmaceutical composition or in separate pharmaceuticalcompositions, such as a tablet, and nivolumab or an antigen bindingfragment thereof or MK-3475 or an antigen binding fragment thereof in avial. When Compounds A and B are not administered simultaneously, thecombination kit can contain Compound B, and/or Compound A in separatepharmaceutical compositions and another pharmaceutical compositioncomprising nivolumab or an antigen binding fragment thereof or MK-3475or an antigen binding fragment thereof, wherein Compound B and/orCompound A are either in a single package or Compound B and/or CompoundA are in separate pharmaceutical compositions in separate packages.

In one embodiment combination kits are provided wherein said compound ofFormula I or a pharmaceutically acceptable salt thereof is provided intablet form suitable for oral administration. In one embodimentcombination kits are provided wherein said compound of Formula II or apharmaceutically acceptable salt thereof is provided in tablet formsuitable for oral administration. In one embodiment combination kits areprovided wherein said anti-PD-1 antibody or antigen binding fragmentthereof is formulated for IV administration. In one embodimentcombination kits are provided wherein said anti-PD-1 antibody or antigenbinding fragment thereof is formulated for subcutaneous administration.

In one aspect there is provided a kit of parts comprising components:

Compound A in association with a pharmaceutically acceptable adjuvant,diluents or carrier; Compound B in association with a pharmaceuticallyacceptable adjuvant, diluents or carrier; and an anti-PD-1 antibody orantigen binding fragment thereof in association with a pharmaceuticallyacceptable adjuvant, diluents or carrier.

In one embodiment of the invention the kit of parts comprise thefollowing components:

Compound B in association with a pharmaceutically acceptable adjuvant,diluents or carrier; and/or

Compound A in association with a pharmaceutically acceptable adjuvant,diluents or carrier;

and an anti-PD-1 antibody or antigen binding fragment thereof, whereinthe components are provided in a form which is suitable for sequential,separate and/or simultaneous administration.

In one embodiment the kit of parts comprises:

a first container comprising Compound B in association with apharmaceutically acceptable adjuvant, diluent or carrier; and/or asecond container comprising Compound A in association with apharmaceutically acceptable adjuvant, diluent or carrier, and a thirdcontainer comprising nivolumab or an antigen binding fragment thereof orMK-3475 or an antigen binding fragment thereof.

The combination kit can also be provided by instruction, such as dosageand administration instructions. Such dosage and administrationinstructions can be of the kind that are provided to a doctor, forexample by a drug product label, or they can be of the kind that areprovided by a doctor, such as instructions to a patient.

The term “loading dose” as used herein will be understood to mean asingle dose or short duration regimen of Compound B or Compound A or ananti-PD-1 antibody having a dosage higher than the maintenance doseadministered to the subject to, for example, rapidly increase the bloodconcentration level of the drug. Suitably, a short duration regimen foruse herein will be from: 1 to 14 days; suitably from 1 to 7 days;suitably from 1 to 3 days; suitably for three days; suitably for twodays; suitably for one day. In some embodiments, the “loading dose” canincrease the blood concentration of the drug to a therapeuticallyeffective level. In some embodiments, the “loading dose” can increasethe blood concentration of the drug to a therapeutically effective levelin conjunction with a maintenance dose of the drug. The “loading dose”of Compound A and/or Compound B can be administered once per day, ormore than once per day (e.g., up to 4 times per day). Suitably the“loading dose” of Compound A and/or Compound B will be administered oncea day. Suitably, the loading dose will be an amount from 2 to 100 timesthe maintenance dose; suitably from 2 to 10 times; suitably from 2 to 5times; suitably 2 times; suitably 3 times; suitably 4 times; suitably 5times. Suitably, the loading dose will be administered for 1 to 7 days;suitably from 1 to 5 days; suitably from 1 to 3 days; suitably for 1day; suitably for 2 days; suitably for 3 days, followed by a maintenancedosing protocol.

The term “maintenance dose” as used herein will be understood to mean adose that is serially administered (for example; at least twice), andwhich is intended to either slowly raise blood concentration levels ofthe compound to a therapeutically effective level, or to maintain such atherapeutically effective level. The maintenance dose for Compound Aand/or Compound B is generally administered once per day and the dailydose of the maintenance dose is lower than the total daily dose of theloading dose.

Suitably the combinations of this invention are administered within a“specified period”.

By the term “specified period” and derivatives thereof, as used hereinis meant the interval of time between the administration of the firstcompound of the combination and last compound of the combination. Forexample, if Compound A is administered first, Compound B second and ananti-PD-1 antibody or antigen binding fragment thereof third, the timeinterval between administration of Compound A and the anti-PD-1 antibodyor antigen binding fragment thereof is the specified period. When onecomponent of the invention is administered more than once a day, thespecified period is calculated based on the first administration of eachcomponent on a specific day. All administrations of a compound of theinvention that are subsequent to the first during a specific day are notconsidered when calculating the specific period.

Suitably, if Compound A, optionally Compound B, and an anti-PD-1antibody or antigen binding fragment thereof are administered within a“specified period” and not administered simultaneously, they are bothadministered within about 24 hours of each other—in this case, thespecified period will be about 24 hours; suitably they will beadministered within about 12 hours of each other—in this case, thespecified period will be about 12 hours; suitably they will beadministered within about 11 hours of each other—in this case, thespecified period will be about 11 hours; suitably they will beadministered within about 10 hours of each other—in this case, thespecified period will be about 10 hours; suitably they will beadministered within about 9 hours of each other—in this case, thespecified period will be about 9 hours; suitably they will beadministered within about 8 hours of each other—in this case, thespecified period will be about 8 hours; suitably they will beadministered within about 7 hours of each other—in this case, thespecified period will be about 7 hours; suitably they will beadministered within about 6 hours of each other—in this case, thespecified period will be about 6 hours; suitably they will beadministered within about 5 hours of each other—in this case, thespecified period will be about 5 hours; suitably they will beadministered within about 4 hours of each other—in this case, thespecified period will be about 4 hours; suitably they will beadministered within about 3 hours of each other—in this case, thespecified period will be about 3 hours; suitably they will beadministered within about 2 hours of each other—in this case, thespecified period will be about 2 hours; suitably they will beadministered within about 1 hour of each other—in this case, thespecified period will be about 1 hour, and is considered simultaneousadministration.

Suitably, when the combination of the invention is administered for a“specified period,” the compounds will be co-administered for a“duration of time”.

By the term “duration of time” and derivatives thereof, when used hereinregarding Compound B and/or Compound A, is meant that Compound A andoptionally Compound B are administered for an indicated number ofconsecutive days, optionally followed by a number of consecutive dayswhere only one of the component compounds is administered.

By the term “duration of time” and derivatives thereof, when used hereinregarding anti-PD-1 antibody or antigen binding fragment thereof, ismeant that the anti-PD-1 antibody or antigen binding fragment thereof isadministered once a week for an indicated number of consecutive weeks.

Regarding “Specified Period” Administration:

Suitably, Compound A, optionally Compound B and an anti-PD-1 antibody orantigen binding fragment thereof will be administered within a specifiedperiod for at least one day—in this case, the duration of time will beat least one day; suitably, during the course of treatment, Compound A,optionally Compound B and the anti-PD-1 antibody or antigen bindingfragment thereof will be administered within a specified period for atleast 3 consecutive days, where the anti-PD-1 antibody or antigenbinding fragment thereof may optionally be administered once during thisperiod—in this case, the duration of time will be at least 3 days;suitably, during the course of treatment, Compound A, optionallyCompound B and an anti-PD-1 antibody or antigen binding fragment thereofwill be administered within a specified period for at least 5consecutive days, where the anti-PD-1 antibody or antigen bindingfragment thereof may optionally be administered once during thisperiod—in this case, the duration of time will be at least 5 days;suitably, during the course of treatment, Compound A, optionallyCompound B and an anti-PD-1 antibody or antigen binding fragment thereofwill be administered within a specified period for at least 7consecutive days, where the anti-PD-1 antibody or antigen bindingfragment thereof may optionally be administered once during thisperiod—in this case, the duration of time will be at least 7 days;suitably, during the course of treatment, Compound A, optionallyCompound B and an anti-PD-1 antibody or antigen binding fragment thereofwill be administered within a specified period for at least 14consecutive days, where the anti-PD-1 antibody or antigen bindingfragment thereof may optionally be administered once a week during thisperiod—in this case, the duration of time will be at least 14 days;suitably, during the course of treatment, Compound A, optionallyCompound B and an anti-PD-1 antibody or antigen binding fragment thereofwill be administered within a specified period for at least 28consecutive days, where the anti-PD-1 antibody or antigen bindingfragment thereof may optionally be administered once a week during thisperiod—in this case, the duration of time will be at least 28 days.

Suitably, if the components are not administered during a “specifiedperiod,” they are administered sequentially. By the term “sequentialadministration,” and derivates thereof, as used herein is meant that thefirst component of the combination of Compound A, optionally Compound Bor an anti-PD-1 antibody or antigen binding fragment thereof isadministered for two or more consecutive days, followed byadministration of a second component in the combination for two or moreconsecutive days, then followed by administration of the last componentin the combination for two or more consecutive days. Also, contemplatedherein is a drug holiday utilized among the sequential administration ofCompound A, optionally Compound B and an anti-PD-1 antibody or antigenbinding fragment thereof. As used herein, a drug holiday is a period ofdays after the sequential administration of one of Compound A,optionally Compound B and an anti-PD-1 antibody or antigen bindingfragment thereof and before the administration of the other component ofthe invention. Suitably the drug holiday will be a period of daysselected from: 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8days, 9 days, 10 days, 11 days, 12 days, 13 days and 14 days.

Regarding Sequential Administration:

Suitably, Compound B will be administered first in the sequence,followed by an optional drug holiday, followed by administration ofCompound A, followed by administration of an anti-PD-1 antibody orantigen binding fragment thereof. Suitably, Compound B is administeredfor 1 to 30 consecutive days, followed by an optional drug holiday,followed by administration of Compound A for 1 to 30 consecutive days,followed by an optional drug holiday, followed by administration of ananti-PD-1 antibody or antigen binding fragment thereof once a week for 1to 10 weeks. Suitably, Compound B is administered for 1 to 21consecutive days, followed by an optional drug holiday, followed byadministration of Compound A for 1 to 21 consecutive days, followed byan optional drug holiday, followed by administration of an anti-PD-1antibody or antigen binding fragment thereof once a week for 1 to 10weeks. Suitably, Compound B is administered for 1 to 14 consecutivedays, followed by an optional drug holiday, followed by administrationof Compound A for 1 to 14 consecutive days, followed by an optional drugholiday, followed by administration of an anti-PD-1 antibody or antigenbinding fragment thereof once a week for 1 to 10 weeks. Suitably,Compound B is administered for 14 consecutive days, followed by anoptional drug holiday, followed by administration of Compound A for 7consecutive days, followed by an optional drug holiday, followed byadministration of an anti-PD-1 antibody or antigen binding fragmentthereof once a week for 1 to 10 weeks. Suitably, Compound B isadministered for 7 consecutive days, followed by an optional drugholiday, followed by administration of Compound A for 7 consecutivedays, followed by an optional drug holiday, followed by administrationof an anti-PD-1 antibody or antigen binding fragment thereof once a weekfor 1 to 10 weeks.

Suitably, Compound A will be administered first in the sequence,followed by an optional drug holiday, followed by optionaladministration of Compound B, followed by administration of an anti-PD-1antibody or antigen binding fragment thereof. Suitably, Compound A isadministered for 1 to 30 consecutive days, followed by an optional drugholiday, followed by optional administration of Compound B for 1 to 30consecutive days, followed by an optional drug holiday, followed byadministration of an anti-PD-1 antibody or antigen binding fragmentthereof once a week for 1 to 10 weeks. Suitably, Compound A isadministered for 1 to 21 consecutive days, followed by an optional drugholiday, followed by optional administration of Compound B for 1 to 21consecutive days, followed by an optional drug holiday, followed byadministration of an anti-PD-1 antibody or antigen binding fragmentthereof once a week for 1 to 10 weeks. Suitably, Compound A isadministered for 1 to 14 consecutive days, followed by an optional drugholiday, followed by optional administration of Compound B for 1 to 14consecutive days, followed by an optional drug holiday, followed byadministration of an anti-PD-1 antibody or antigen binding fragmentthereof once a week for 1 to 10 weeks. Suitably, Compound A isadministered for 14 consecutive days, followed by an optional drugholiday, followed by optional administration of Compound B for 14consecutive days, followed by an optional drug holiday, followed byadministration of an anti-PD-1 antibody or antigen binding fragmentthereof once a week for 1 to 10 weeks. Suitably, Compound A isadministered for 7 consecutive days, followed by an optional drugholiday, followed by optional administration of Compound B for 7consecutive days, followed by an optional drug holiday, followed byadministration of an anti-PD-1 antibody or antigen binding fragmentthereof once a week for 1 to 10 weeks.

Suitably, an anti-PD-1 antibody or antigen binding fragment thereof willbe administered first in the sequence, followed by an optional drugholiday, followed by optional administration of Compound B, followed byan optional drug holiday, followed by administration of Compound A.Suitably, an anti-PD-1 antibody or antigen binding fragment thereof isadministered once a week for from 1-10 weeks, followed by an optionaldrug holiday, followed by optional administration of Compound B for 1 to30 consecutive days, followed by an optional drug holiday, followed byadministration of Compound A for 1 to 30 consecutive days. Suitably, ananti-PD-1 antibody or antigen binding fragment thereof is administeredonce a week for from 1-10 weeks, followed by an optional drug holiday,followed by optional administration of Compound B for 1 to 21consecutive days, followed by an optional drug holiday, followed byadministration of Compound A for 1 to 21 consecutive days. Suitably, ananti-PD-1 antibody or antigen binding fragment thereof is administeredonce a week for from 1-10 weeks, followed by an optional drug holiday,followed by optional administration of Compound B for 1 to 14consecutive days, followed by an optional drug holiday, followed byadministration of Compound A for 1 to 14 consecutive days. Suitably, ananti-PD-1 antibody or antigen binding fragment thereof is administeredonce a week for from 1-10 weeks, followed by an optional drug holiday,followed by optional administration of Compound B for 14 consecutivedays, followed by an optional drug holiday, followed by administrationof Compound A for 14 consecutive days. Suitably, an anti-PD-1 antibodyor antigen binding fragment thereof is administered once a week for from1-10 weeks, followed by an optional drug holiday, followed by optionaladministration of Compound B for 7 consecutive days, followed by anoptional drug holiday, followed by administration of Compound A for 7consecutive days.

Suitably, an anti-PD-1 antibody or antigen binding fragment thereof willbe administered first in the sequence, followed by an optional drugholiday, followed by administration of Compound A, followed by anoptional drug holiday, followed by optional administration of CompoundB. Suitably, an anti-PD-1 antibody or antigen binding fragment thereofis administered once a week for 1-10 weeks, followed by an optional drugholiday, followed by administration of Compound A for 1 to 30consecutive days, followed by an optional drug holiday, followed byoptional administration of Compound B for 1 to 30 consecutive days.Suitably, an anti-PD-1 antibody or antigen binding fragment thereof isadministered once a week for from 1-10 weeks, followed by an optionaldrug holiday, followed by administration of Compound A for 1 to 21consecutive days, followed by an optional drug holiday, followed byoptional administration of Compound B for 1 to 21 consecutive days.Suitably, an anti-PD-1 antibody or antigen binding fragment thereof isadministered once a week for 1-10 weeks, followed by an optional drugholiday, followed by administration of Compound A for 1 to 14consecutive days, followed by an optional drug holiday, followed byoptional administration of Compound B for 1 to 14 consecutive days.Suitably, an anti-PD-1 antibody or antigen binding fragment thereof isadministered once a week for from 1-10 weeks, followed by an optionaldrug holiday, followed by administration of Compound A for 14consecutive days, followed by an optional drug holiday, followed byoptional administration of Compound B for 14 consecutive days. Suitably,an anti-PD-1 antibody or antigen binding fragment thereof isadministered once a week for from 1-10 weeks, followed by an optionaldrug holiday, followed by administration of Compound A for 7 consecutivedays, followed by an optional drug holiday, followed by optionaladministration of Compound B for 7 consecutive days.

Suitably, Compound A will be administered first in the sequence,followed by an optional drug holiday, followed by administration of ananti-PD-1 antibody or antigen binding fragment thereof, followed byoptional administration of Compound B. Suitably, Compound A isadministered for 1 to 30 consecutive days, followed by an optional drugholiday, followed by administration of an anti-PD-1 antibody or antigenbinding fragment thereof once a week for 1 to 10 weeks, followed by anoptional drug holiday, followed by optional administration of Compound Bfor 1 to 30 consecutive days. Suitably, Compound A is administered for 1to 21 consecutive days, followed by an optional drug holiday, followedby administration of an anti-PD-1 antibody or antigen binding fragmentthereof once a week for 1 to 10 weeks, followed by an optional drugholiday, followed by optional administration of Compound B for 1 to 21consecutive days. Suitably, Compound A is administered for 1 to 14consecutive days, followed by an optional drug holiday, followed byadministration of an anti-PD-1 antibody or antigen binding fragmentthereof once a week for 1 to 10 weeks, followed by an optional drugholiday, followed by optional administration of Compound B for 1 to 14consecutive days. Suitably, Compound A is administered for 14consecutive days, followed by an optional drug holiday, followed byadministration of an anti-PD-1 antibody or antigen binding fragmentthereof once a week for 1 to 10 weeks, followed by an optional drugholiday, followed by optional administration of Compound B for 14consecutive days. Suitably, Compound A is administered for 7 consecutivedays, followed by an optional drug holiday, followed by administrationof an anti-PD-1 antibody or antigen binding fragment thereof once a weekfor 1 to 10 weeks, followed by an optional drug holiday, followed byoptional administration of Compound B for 7 consecutive days.

Suitably, Compound B will be administered first in the sequence,followed by an optional drug holiday, followed by administration of ananti-PD-1 antibody or antigen binding fragment thereof, followed byadministration of Compound A. Suitably, Compound B is administered for 1to 30 consecutive days, followed by an optional drug holiday, followedby administration of an anti-PD-1 antibody or antigen binding fragmentthereof once a week for 1 to 10 weeks, followed by an optional drugholiday, followed by administration of Compound A for 1 to 30consecutive days. Suitably, Compound B is administered for 1 to 21consecutive days, followed by an optional drug holiday, followed byadministration of an anti-PD-1 antibody or antigen binding fragmentthereof once a week for 1 to 10 weeks, followed by an optional drugholiday, followed by administration of Compound A for 1 to 21consecutive days. Suitably, Compound B is administered for 1 to 14consecutive days, followed by an optional drug holiday, followed byadministration of an anti-PD-1 antibody or antigen binding fragmentthereof once a week for 1 to 10 weeks, followed by an optional drugholiday, followed by administration of Compound A for 1 to 14consecutive days. Suitably, Compound B is administered for 14consecutive days, followed by an optional drug holiday, followed byadministration of an anti-PD-1 antibody or antigen binding fragmentthereof once a week for 1 to 10 weeks, followed by an optional drugholiday, followed by administration of Compound A for 14 consecutivedays. Suitably, Compound B is administered for 7 consecutive days,followed by an optional drug holiday, followed by administration of ananti-PD-1 antibody or antigen binding fragment thereof once a week for 1to 10 weeks, followed by an optional drug holiday, followed byadministration of Compound A for 7 consecutive days.

It is understood that a “specified period” administration and a“sequential” administration can be followed by repeat dosing or can befollowed by an alternate dosing protocol, and a drug holiday may precedethe repeat dosing or alternate dosing protocol.

Suitably, the amount of Compound A (based on weight ofunsalted/unsolvated amount) administered as part of the combinationaccording to the present invention will be an amount selected from about0.125 mg to about 10 mg; suitably, the amount will be selected fromabout 0.25 mg to about 9 mg; suitably, the amount will be selected fromabout 0.25 mg to about 8 mg; suitably, the amount will be selected fromabout 0.5 mg to about 8 mg; suitably, the amount will be selected fromabout 0.5 mg to about 7 mg; suitably, the amount will be selected fromabout 1 mg to about 7 mg; suitably, the amount will be about 5 mg.Accordingly, the amount of Compound A administered as part of thecombination according to the present invention will be an amountselected from about 0.125 mg to about 10 mg. For example, the amount ofCompound A administered as part of the combination according to thepresent invention can be 0.125 mg, 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.5mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg.

Suitably, the selected amount of Compound A is administered from 1 to 4times a day. Suitably, the selected amount of Compound A is administeredtwice a day. Suitably, the selected amount of Compound A is administeredonce a day. Suitably, the administration of Compound A will begin as aloading dose. Suitably, the loading dose will be an amount from 2 to 100times the maintenance dose; suitably from 2 to 10 times; suitably from 2to 5 times; suitably 2 times; suitably 3 times; suitably 4 times;suitably 5 times. Suitably, the loading does will be administered from 1to 7 days; suitably from 1 to 5 days; suitably from 1 to 3 days;suitably for 1 day; suitably for 2 days; suitably for 3 days, followedby a maintenance dosing protocol.

Suitably, the amount of Compound B (based on weight ofunsalted/unsolvated amount) optionally administered as part of thecombination according to the present invention will be an amountselected from about 10 mg to about 600 mg. Suitably, the amount will beselected from about 30 mg to about 300 mg; suitably, the amount will beselected from about 30 mg to about 280 mg; suitably, the amount will beselected from about 40 mg to about 260 mg; suitably, the amount will beselected from about 60 mg to about 240 mg; suitably, the amount will beselected from about 80 mg to about 220 mg; suitably, the amount will beselected from about 90 mg to about 210 mg; suitably, the amount will beselected from about 100 mg to about 200 mg, suitably, the amount will beselected from about 110 mg to about 190 mg, suitably, the amount will beselected from about 120 mg to about 180 mg, suitably, the amount will beselected from about 130 mg to about 170 mg, suitably, the amount will beselected from about 140 mg to about 160 mg, suitably, the amount will be150 mg. Accordingly, the amount of Compound B administered as part ofthe combination according to the present invention will be an amountselected from about 10 mg to about 300 mg. For example, the amount ofCompound B administered as part of the combination according to thepresent invention is suitably selected from 10 mg, 20 mg, 30 mg, 40 mg,50 mg, 60 mg, 70 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg,115 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg,160 mg, 165 mg, 170 mg, 175 mg, 180 mg, 185 mg, 190 mg, 195 mg, 200 mg,205 mg, 210 mg, 215 mg, 220 mg, 225 mg, 230 mg, 235 mg, 240 mg, 245 mg,250 mg, 255 mg, 260 mg, 265 mg, 270 mg, 275 mg, 280 mg, 285 mg, 290 mg,295 mg and 300 mg. Suitably, the selected amount of Compound B isadministered from 1 to 4 times a day. Suitably, the selected amount ofCompound B is administered twice a day. Suitably, Compound B isadministered twice a day. Suitably, the selected amount of Compound B isadministered once a day.

Suitably, the administration of Compound B will begin as a loading dose.Suitably, the loading dose will be an amount from 2 to 100 times themaintenance dose; suitably from 2 to 10 times; suitably from 2 to 5times; suitably 2 times; suitably 3 times; suitably 4 times; suitably 5times. Suitably, the loading does will be administered from 1 to 7 days;suitably from 1 to 5 days; suitably from 1 to 3 days; suitably for 1day; suitably for 2 days; suitably for 3 days, followed by a maintenancedosing protocol.

An anti-PD-1 antibody or antigen binding fragment thereof isadministered at a dosage amount of from about 50 mg/m²/week to about 700mg/m²/week; suitably, from 100 mg/m²/week to about 600 mg/m²/week;suitably, from 200 mg/m²/week to about 500 mg/m²/week or at a dose of 1,2, 3, 5 or 10 mg/kg at intervals of about 14 days (±2 days) or about 21days (±2 days) or about 30 days (±2 days) throughout the course oftreatment.

In an embodiment, an anti-PD-1 antibody or antigen binding fragmentthereof is administered once a week with initial administration being inan amount of from 400 mg/m²/week to about 500 mg/m²/week and eachsubsequent administration being in an amount of from 200 mg/m²/week to300 mg/m²/week. In one embodiment, an anti-PD-1 antibody is administeredat a dose of about 0.3 to about 3.0 mg/kg up to about 10 mg/kg.Anti-PD-1 antibodies or antigen binding fragments thereof of the presentinvention can be formulation for intravenous administration and/orsubcutaneous administration. Doses can be administered daily, weekly,once every two weeks, once every three weeks, once every four weeks,and/or monthly.

The present invention also provides a medicament which comprises a PD-1antagonist as described above and a pharmaceutically acceptableexcipient. When the PD-1 antagonist is a biotherapeutic agent, e.g., amAb, the antagonist may be produced in CHO cells using conventional cellculture and recovery/purification technologies.

In some embodiments, a medicament comprising an anti-PD-1 antibody asthe PD-1 antagonist may be provided as a liquid formulation or preparedby reconstituting a lyophilized powder with sterile water for injectionprior to use. WO 2012/135408 describes the preparation of liquid andlyophilized medicaments comprising MK-3475 that are suitable for use inthe present invention. In some preferred embodiments, a medicamentcomprising MK-3475 is provided in a glass vial which contains about 50mg of MK-3475.

One embodiment of the present invention provides a combination ofCompound A, administered once a day; Compound B, optionally administeredonce or twice a day; and MK-3475 administered according to theaforementioned protocol, for a period of at least 8 weeks, suitably fora period of at least 4 weeks, suitably for a period of at least 2 weeks,suitably for a period of at least 10 days, suitably for a period of atleast 7 days, suitably all three compounds are administered on the firstday of each 7 day period.

One embodiment of the present invention provides a combination ofCompound A, administered once a day; Compound B, optionally administeredonce or twice a day; and nivolumab administered according to theaforementioned protocol, for a period of at least 8 weeks, suitably fora period of at least 4 weeks, suitably for a period of at least 2 weeks,suitably for a period of at least 10 days, suitably for a period of atleast 7 days, suitably all three compounds are administered on the firstday of each 7 day period.

As used herein, all amounts specified for Compound A and Compound B areindicated as the amount of free or unsalted compound.

In some embodiments of any of the above pharmaceutical compositions,combinations, combination kits, uses and treatment methods, Compound Ais administered once daily as a 2-mg MEKINIST (trametinib) tablet,Compound B is administered twice daily as two 75-mg or three 50-mgTANIFLAR (dabrafenib) capsules, and the PD-1 antagonist is MK-3475,which is administered by intravenous infusion once every three weeks ata dose of 2 mg/kg or once every 2 weeks at a dose of 10 mg/kg.

Method of Treatment

The combinations and combination therapies of the invention are believedto have utility in disorders wherein the inhibition of BRAF and/or MEKand/or enhancement of the immune response through blocking theinhibitory signal of PD-1 is beneficial.

The present invention thus also provides a combination of the invention,for use in therapy, particularly in the treatment of disorders whereinthe inhibition of BRAF and/or MEK and/or enhancing the immune responseby blocking the inhibitory signaling of PD-1 is beneficial, particularlycancer.

A further aspect of the invention provides a method of treatment of adisorder wherein inhibition of BRAF and/or MEK and/or enhancing theimmune response by blocking the inhibitory signaling of PD-1 isbeneficial, comprising administering a combination of the invention.

A further aspect of the present invention provides the use of acombination of the invention in the manufacture of a medicament for thetreatment of a disorder wherein the inhibition of BRAF and/or MEK and/orenhancing the immune response by blocking the inhibitory signaling ofPD-1 is beneficial.

Typically, the disorder is a cancer such that inhibition of BRAF and/orMEK and/or enhancing the immune response by blocking the inhibitorysignaling of PD-1 has a beneficial effect. Examples of cancers that aresuitable for treatment with combination of the invention include, butare limited to, both primary and metastatic forms of head and neck,breast, lung, colon, ovary, and prostate cancers. Suitably the cancer isselected from: brain (gliomas), glioblastomas, astrocytomas,glioblastoma multiforme, Bannayan-Zonana syndrome, Cowden disease,Lhermitte-Duclos disease, breast, inflammatory breast cancer, Wilm'stumor, Ewing's sarcoma, Rhabdomyosarcoma, ependymoma, medulloblastoma,colon, head and neck, kidney, lung, liver, melanoma, ovarian,pancreatic, prostate, sarcoma, osteosarcoma, giant cell tumor of bone,thyroid, lymphoblastic T cell leukemia, Chronic myelogenous leukemia,Chronic lymphocytic leukemia, Hairy-cell leukemia, acute lymphoblasticleukemia, acute myelogenous leukemia, AML, Chronic neutrophilicleukemia, Acute lymphoblastic T cell leukemia, plasmacytoma,Immunoblastic large cell leukemia, Mantle cell leukemia, Multiplemyeloma Megakaryoblastic leukemia, multiple myeloma, acutemegakaryocytic leukemia, promyelocytic leukemia, Erythroleukemia,malignant lymphoma, hodgkins lymphoma, non-hodgkins lymphoma,lymphoblastic T cell lymphoma, Burkitt's lymphoma, follicular lymphoma,neuroblastoma, bladder cancer, urothelial cancer, lung cancer, vulvalcancer, cervical cancer, endometrial cancer, renal cancer, mesothelioma,esophageal cancer, salivary gland cancer, hepatocellular cancer, gastriccancer, nasopharangeal cancer, buccal cancer, cancer of the mouth, GIST(gastrointestinal stromal tumor) and testicular cancer.

Additionally, examples of a cancer to be treated include Barret'sadenocarcinoma; billiary tract carcinomas; breast cancer; cervicalcancer; cholangiocarcinoma; central nervous system tumors includingprimary CNS tumors such as glioblastomas, astrocytomas (e.g.,glioblastoma multiforme) and ependymomas, and secondary CNS tumors(i.e., metastases to the central nervous system of tumors originatingoutside of the central nervous system); colorectal cancer includinglarge intestinal colon carcinoma; gastric cancer; carcinoma of the headand neck including squamous cell carcinoma of the head and neck;hematologic cancers including leukemias and lymphomas such as acutelymphoblastic leukemia, acute myelogenous leukemia (AML),myelodysplastic syndromes, chronic myelogenous leukemia, Hodgkin'slymphoma, non-Hodgkin's lymphoma, megakaryoblastic leukemia, multiplemyeloma and erythroleukemia; hepatocellular carcinoma; lung cancerincluding small cell lung cancer and non-small cell lung cancer; ovariancancer; endometrial cancer; pancreatic cancer; pituitary adenoma;prostate cancer; renal cancer; sarcoma; skin cancers includingmelanomas; and thyroid cancers.

Suitably, the present invention relates to a method for treating orlessening the severity of a cancer selected from: brain (gliomas),glioblastomas, astrocytomas, glioblastoma multiforme, Bannayan-Zonanasyndrome, Cowden disease, Lhermitte-Duclos disease, breast, colon, headand neck, kidney, lung, liver, melanoma, ovarian, pancreatic, prostate,sarcoma and thyroid.

Suitably, the present invention relates to a method for treating orlessening the severity of a cancer selected from ovarian, breast,pancreatic and prostate.

Suitably the present invention relates to a method for treating orlessening the severity of pre-cancerous syndromes in a mammal, includinga human, wherein the pre-cancerous syndrome is selected from: cervicalintraepithelial neoplasia, monoclonal gammapathy of unknown significance(MGUS), myelodysplastic syndrome, aplastic anemia, cervical lesions,skin nevi (pre-melanoma), prostatic intraepithleial (intraductal)neoplasia (PIN), Ductal Carcinoma in situ (DCIS), colon polyps andsevere hepatitis or cirrhosis.

Suitably, the present invention relates to a method of treating orlessening the severity of a cancer that is either wild type or mutantfor Raf and KRAS and either wild type or mutant for PI3K/Pten. Thisincludes patients with a cancer that is wild type for both Raf, KRAS,and PI3K/PTEN, mutant for Raf, KRAS and PI3K/PTEN, mutant for Raf andwild type for KRAS and PI3K/PTEN and wild type for Raf and KRAS andmutant for PI3K/PTEN.

The term “wild type” as is understood in the art refers to a polypeptideor polynucleotide sequence that occurs in a native population withoutgenetic modification. As is also understood in the art, a “mutant”includes a polypeptide or polynucleotide sequence having at least onemodification to an amino acid or nucleic acid compared to thecorresponding amino acid or nucleic acid found in a wild typepolypeptide or polynucleotide, respectively. Included in the term mutantis Single Nucleotide Polymorphism (SNP) where a single base pairdistinction exists in the sequence of a nucleic acid strand compared tothe most prevalently found (wild type) nucleic acid strand. Suitably,when the cancer is wild type for Raf or BRAF wild type or tests negativefor a BRAF V600 mutation, only Compound A and an anti-PD-1 antibody orantigen binding fragment thereof are administered, and Compound B is notadministered.

Cancers that are either wild type or mutant for Raf, either wild type ormutant for PI3K/Pten, and either wild type or mutant are identified byknown methods.

For example, wild type or mutant Raf or PI3K/PTEN tumor cells can beidentified by DNA amplification and sequencing techniques, DNA and RNAdetection techniques, including, but not limited to Northern andSouthern blot, respectively, and/or various biochip and arraytechnologies. Wild type and mutant polypeptides can be detected by avariety of techniques including, but not limited to immunodiagnostictechniques such as ELISA, Western blot or immunocytochemistry. Suitably,Pyrophosphorolysis-activated polymerization (PAP) and/or PCR methods maybe used. Liu, Q et al; Human Mutation 23:426-436 (2004).

In one preferred embodiment, a patient with advanced or metastaticmelanoma that tests positive for a BRAF V600 mutation is administered acombination of: (i) MK-3475 at a dose of 1 mg/kg Q3W, 2 mg/kg Q3W or 10mg/kg Q2W; (ii) dabrafenib at a dose of 100 BID or 150 BID and (iii)trametinib at a dose of 1 mg or 2 mg QD.

In another preferred embodiment, a patient with advanced or metastaticmelanoma that tests negative for a BRAF V600 mutation is administered acombination of: (i) MK-3475 at a dose of 2 mg/kg Q3W or 10 mg/kg Q2W andtrametinib at a dose of 1 mg QD or 2 mg QD.

In yet another preferred embodiment, a patient with advanced ormetastatic melanoma that tests positive for a BRAF V600 mutation isadministered a combination of: (i) MK-3475 at a dose of 1 mg/kg Q3W, 2mg/kg Q3W or 10 mg/kg Q2W and dabrafenib at a dose of 100 mg BID or 150BID.

In any of the above preferred embodiments, the preferred starting dosesare: MK-3475 at 2 mg/kg Q3W, dabrafenib at 150 mg BID and trametinib at2 mg QD. If the patient experiences a kinase-inhibitor related adverseevent during treatment with the combination, the dose of dabrafeniband/or trametinib is preferably reduced by up to one half the startingdose. If the patient experiences an immune related adverse event duringtreatment with the combination, the MK-3475 dose is reduced to one-halfof the starting dose, e.g., 1 mg/kg Q3W.

In each of the above preferred embodiments for the treatment of advancedor metastatic melanoma, MK-3475 is preferably administered as a liquidpharmaceutical composition which comprises 25 mg/ml MK-3475, 7% (w/v)sucrose, 0.02% (w/v) polysorbate 80 in 10 mM histidine buffer pH 5.5,and the selected dose of the composition is administered by IV infusionover a time period of about 30 minutes (e.g., 25 to 40 minutes).

In any of the above embodiments for the treatment of advanced ormetastatic melanoma, the combination is administered for at least onecycle of six weeks, and preferably the treatment duration is any of: atleast 6, 12, 18 or 24 months, at least 2 cycles after a CR, andobservation of PD.

In any of the above embodiments for the treatment of advanced ormetastatic melanoma, the combination is administered to patients with atumor that tests positive for PD-L1 expression.

In any of the above embodiments for treatment of advanced or metastaticmelanoma, the patient preferably does not have a diagnosis of uveal orocular melanoma, and preferably has not received prior systemic therapyfor metastatic or advance melanoma with an agent targeting PD-1, PD-L1,CTLA4, BRAF, MEK or other molecules in the MAPK pathway.

The combination of the invention may be used alone or in combinationwith one or more other therapeutic agents. The invention thus providesin a further aspect a further combination comprising a combination ofthe invention with a further therapeutic agent or agents, compositionsand medicaments comprising the combination and use of the furthercombination, compositions and medicaments in therapy, in particular inthe treatment of diseases susceptible to inhibition of MEK and/orinhibition of BRAF and/or an enhanced immune response by blocking theinhibitory signaling of PD-1.

In one embodiment, a combination of the invention may be employed withother therapeutic methods of cancer treatment. In particular, inanti-neoplastic therapy, combination therapy with otherchemotherapeutic, hormonal, antibody agents as well as surgical and/orradiation treatments other than those mentioned above are envisaged.Combination therapies according to the present invention thus includethe administration of Compound A, Compound B and MK-3475 or nivolumab aswell as optional use of other therapeutic agents including otheranti-neoplastic agents. Such combination of agents may be administeredtogether or separately and, when administered separately this may occursimultaneously or sequentially in any order, both close and remote intime. In one embodiment, the pharmaceutical combination includesCompound A, Compound B and MK-3475, and optionally at least oneadditional anti-neoplastic agent. In one embodiment, the pharmaceuticalcombination includes Compound A, Compound B and nivolumab, andoptionally at least one additional anti-neoplastic agent.

In one embodiment, the further anti-cancer therapy is surgical and/orradiotherapy.

In one embodiment, the further anti-cancer therapy is at least oneadditional anti-neoplastic agent.

Any anti-neoplastic agent that has activity versus a susceptible tumorbeing treated may be utilized in the combination. Typicalanti-neoplastic agents useful include, but are not limited to,anti-microtubule agents such as diterpenoids and vinca alkaloids;platinum coordination complexes; alkylating agents such as nitrogenmustards, oxazaphosphorines, alkylsulfonates, nitrosoureas, andtriazenes; antibiotic agents such as anthracyclins, actinomycins andbleomycins; topoisomerase II inhibitors such as epipodophyllotoxins;antimetabolites such as purine and pyrimidine analogues and anti-folatecompounds; topoisomerase I inhibitors such as camptothecins; hormonesand hormonal analogues; signal transduction pathway inhibitors;non-receptor tyrosine angiogenesis inhibitors; immunotherapeutic agents;proapoptotic agents; and cell cycle signaling inhibitors.

Anti-microtubule or anti-mitotic agents: Anti-microtubule oranti-mitotic agents are phase specific agents active against themicrotubules of tumor cells during M or the mitosis phase of the cellcycle. Examples of anti-microtubule agents include, but are not limitedto, diterpenoids and vinca alkaloids.

Diterpenoids, which are derived from natural sources, are phase specificanti-cancer agents that operate at the G₂/M phases of the cell cycle. Itis believed that the diterpenoids stabilize the β-tubulin subunit of themicrotubules, by binding with this protein. Disassembly of the proteinappears then to be inhibited with mitosis being arrested and cell deathfollowing. Examples of diterpenoids include, but are not limited to,paclitaxel and its analog docetaxel.

Paclitaxel, 5β,20-epoxy-1,2α,4,7β,10β,13α-hexa-hydroxytax-11-en-9-one4,10-diacetate 2-benzoate 13-ester with(2R,3S)—N-benzoyl-3-phenylisoserine; is a natural diterpene productisolated from the Pacific yew tree Taxus brevifolia and is commerciallyavailable as an injectable solution TAXOL®. It is a member of the taxanefamily of terpenes. Paclitaxel has been approved for clinical use in thetreatment of refractory ovarian cancer in the United States (Markman etal., Yale Journal of Biology and Medicine, 64:583, 1991; McGuire et al.,Ann. Intern, Med., 111:273, 1989) and for the treatment of breast cancer(Holmes et al., J. Nat. Cancer Inst., 83:1797, 1991.) It is a potentialcandidate for treatment of neoplasms in the skin (Einzig et. al., Proc.Am. Soc. Clin. Oncol., 20:46) and head and neck carcinomas (Forastireet. al., Sem. Oncol., 20:56, 1990). The compound also shows potentialfor the treatment of polycystic kidney disease (Woo et. al., Nature,368:750. 1994), lung cancer and malaria. Treatment of patients withpaclitaxel results in bone marrow suppression (multiple cell lineages,Ignoff, R. J. et. al, Cancer Chemotherapy Pocket Guide, 1998) related tothe duration of dosing above a threshold concentration (50 nM) (Kearns,C. M. et. al., Seminars in Oncology, 3(6) p. 16-23, 1995).

Docetaxel, (2R,3S)—N-carboxy-3-phenylisoserine,N-tert-butyl ester,13-ester with 5β-20-epoxy-1,2α,4,7β,10β,13α-hexahydroxytax-11-en-9-one4-acetate 2-benzoate, trihydrate; is commercially available as aninjectable solution as TAXOTERE®. Docetaxel is indicated for thetreatment of breast cancer. Docetaxel is a semisynthetic derivative ofpaclitaxel q.v., prepared using a natural precursor,10-deacetyl-baccatin III, extracted from the needle of the European Yewtree.

Vinca alkaloids are phase specific anti-neoplastic agents derived fromthe periwinkle plant. Vinca alkaloids act at the M phase (mitosis) ofthe cell cycle by binding specifically to tubulin. Consequently, thebound tubulin molecule is unable to polymerize into microtubules.Mitosis is believed to be arrested in metaphase with cell deathfollowing. Examples of vinca alkaloids include, but are not limited to,vinblastine, vincristine, and vinorelbine.

Vinblastine, vincaleukoblastine sulfate, is commercially available asVELBAN® as an injectable solution. Although, it has possible indicationas a second line therapy of various solid tumors, it is primarilyindicated in the treatment of testicular cancer and various lymphomasincluding Hodgkin's Disease; and lymphocytic and histiocytic lymphomas.Myelosuppression is the dose limiting side effect of vinblastine.

Vincristine, vincaleukoblastine, 22-oxo-, sulfate, is commerciallyavailable as ONCOVIN® as an injectable solution. Vincristine isindicated for the treatment of acute leukemias and has also found use intreatment regimens for Hodgkin's and non-Hodgkin's malignant lymphomas.Alopecia and neurologic effects are the most common side effect ofvincristine and to a lesser extent myelosupression and gastrointestinalmucositis effects occur.

Vinorelbine,3′,4′-didehydro-4′-deoxy-C′-norvincaleukoblastine[R—(R*,R*)-2,3-dihydroxybutanedioate(1:2)(salt)], commercially available as an injectable solution ofvinorelbine tartrate (NAVELBINE®), is a semisynthetic vinca alkaloid.Vinorelbine is indicated as a single agent or in combination with otherchemotherapeutic agents, such as cisplatin, in the treatment of varioussolid tumors, particularly non-small cell lung, advanced breast, andhormone refractory prostate cancers. Myelosuppression is the most commondose limiting side effect of vinorelbine.

Platinum coordination complexes: Platinum coordination complexes arenon-phase specific anti-cancer agents, which are interactive with DNA.The platinum complexes enter tumor cells, undergo, aquation and formintra- and interstrand crosslinks with DNA causing adverse biologicaleffects to the tumor. Examples of platinum coordination complexesinclude, but are not limited to, oxaliplatin, cisplatin and carboplatin.

Cisplatin, cis-diamminedichloroplatinum, is commercially available asPLATINOL® as an injectable solution. Cisplatin is primarily indicated inthe treatment of metastatic testicular and ovarian cancer and advancedbladder cancer.

Carboplatin, platinum, diammine[1,1-cyclobutane-dicarboxylate(2+O,O′],is commercially available as PARAPLATIN® as an injectable solution.Carboplatin is primarily indicated in the first and second linetreatment of advanced ovarian carcinoma.

Alkylating agents: Alkylating agents are non-phase anti-cancer specificagents and strong electrophiles. Typically, alkylating agents formcovalent linkages, by alkylation, to DNA through nucleophilic moietiesof the DNA molecule such as phosphate, amino, sulfhydryl, hydroxyl,carboxyl, and imidazole groups. Such alkylation disrupts nucleic acidfunction leading to cell death. Examples of alkylating agents include,but are not limited to, nitrogen mustards such as cyclophosphamide,melphalan, and chlorambucil; alkyl sulfonates such as busulfan;nitrosoureas such as carmustine; and triazenes such as dacarbazine.

Cyclophosphamide,2-[bis(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxidemonohydrate, is commercially available as an injectable solution ortablets as CYTOXAN®. Cyclophosphamide is indicated as a single agent orin combination with other chemotherapeutic agents, in the treatment ofmalignant lymphomas, multiple myeloma, and leukemias.

Melphalan, 4-[bis(2-chloroethyl)amino]-L-phenylalanine, is commerciallyavailable as an injectable solution or tablets as ALKERAN®. Melphalan isindicated for the palliative treatment of multiple myeloma andnon-resectable epithelial carcinoma of the ovary. Bone marrowsuppression is the most common dose limiting side effect of melphalan.

Chlorambucil, 4-[bis(2-chloroethyl)amino]benzenebutanoic acid, iscommercially available as LEUKERAN® tablets. Chlorambucil is indicatedfor the palliative treatment of chronic lymphatic leukemia, andmalignant lymphomas such as lymphosarcoma, giant follicular lymphoma,and Hodgkin's disease.

Busulfan, 1,4-butanediol dimethanesulfonate, is commercially availableas MYLERAN® TABLETS. Busulfan is indicated for the palliative treatmentof chronic myelogenous leukemia.

Carmustine, 1,3-[bis(2-chloroethyl)-1-nitrosourea, is commerciallyavailable as single vials of lyophilized material as BiCNU®. Carmustineis indicated for the palliative treatment as a single agent or incombination with other agents for brain tumors, multiple myeloma,Hodgkin's disease, and non-Hodgkin's lymphomas.

Dacarbazine, 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide, iscommercially available as single vials of material as DTIC-Dome®.Dacarbazine is indicated for the treatment of metastatic malignantmelanoma and in combination with other agents for the second linetreatment of Hodgkin's Disease.

Antibiotic anti-neoplastics: Antibiotic anti-neoplastics are non-phasespecific agents, which bind or intercalate with DNA. Typically, suchaction results in stable DNA complexes or strand breakage, whichdisrupts ordinary function of the nucleic acids leading to cell death.Examples of antibiotic anti-neoplastic agents include, but are notlimited to, actinomycins such as dactinomycin, anthrocyclins such asdaunorubicin and doxorubicin; and bleomycins.

Dactinomycin, also know as Actinomycin D, is commercially available ininjectable form as COSMEGEN®. Dactinomycin is indicated for thetreatment of Wilm's tumor and rhabdomyosarcoma.

Daunorubicin,(8S-cis-)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12naphthacenedione hydrochloride, is commercially available as a liposomalinjectable form as DAUNOXOME® or as an injectable as CERUBIDINE®.Daunorubicin is indicated for remission induction in the treatment ofacute nonlymphocytic leukemia and advanced HIV associated Kaposi'ssarcoma.

Doxorubicin,(8S,10S)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)oxy]-8-glycoloyl,7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12 naphthacenedionehydrochloride, is commercially available as an injectable form as RUBEX®or ADRIAMYCIN RDF®. Doxorubicin is primarily indicated for the treatmentof acute lymphoblastic leukemia and acute myeloblastic leukemia, but isalso a useful component in the treatment of some solid tumors andlymphomas.

Bleomycin, a mixture of cytotoxic glycopeptide antibiotics isolated froma strain of Streptomyces verticillus, is commercially available asBLENOXANE®. Bleomycin is indicated as a palliative treatment, as asingle agent or in combination with other agents, of squamous cellcarcinoma, lymphomas, and testicular carcinomas.

Topoisomerase II inhibitors: Topoisomerase II inhibitors include, butare not limited to, epipodophyllotoxins.

Epipodophyllotoxins are phase specific anti-neoplastic agents derivedfrom the mandrake plant. Epipodophyllotoxins typically affect cells inthe S and G₂ phases of the cell cycle by forming a ternary complex withtopoisomerase II and DNA causing DNA strand breaks. The strand breaksaccumulate and cell death follows. Examples of epipodophyllotoxinsinclude, but are not limited to, etoposide and teniposide.

Etoposide, 4′-demethyl-epipodophyllotoxin9[4,6-0-(R)-ethylidene-β-D-glucopyranoside], is commercially availableas an injectable solution or capsules as VePESID® and is commonly knownas VP-16. Etoposide is indicated as a single agent or in combinationwith other chemotherapy agents in the treatment of testicular andnon-small cell lung cancers.

Teniposide, 4′-demethyl-epipodophyllotoxin9[4,6-0-(R)-thenylidene-β-D-glucopyranoside], is commercially availableas an injectable solution as VUMON® and is commonly known as VM-26.Teniposide is indicated as a single agent or in combination with otherchemotherapy agents in the treatment of acute leukemia in children.

Antimetabolite neoplastic agents: Antimetabolite neoplastic agents arephase specific anti-neoplastic agents that act at S phase (DNAsynthesis) of the cell cycle by inhibiting DNA synthesis or byinhibiting purine or pyrimidine base synthesis and thereby limiting DNAsynthesis. Consequently, S phase does not proceed and cell deathfollows. Examples of antimetabolite anti-neoplastic agents include, butare not limited to, fluorouracil, methotrexate, cytarabine,mecaptopurine, thioguanine, and gemcitabine.

5-fluorouracil, 5-fluoro-2,4-(1H,3H)pyrimidinedione, is commerciallyavailable as fluorouracil. Administration of 5-fluorouracil leads toinhibition of thymidylate synthesis and is also incorporated into bothRNA and DNA. The result typically is cell death. 5-fluorouracil isindicated as a single agent or in combination with other chemotherapyagents in the treatment of carcinomas of the breast, colon, rectum,stomach and pancreas. Other fluoropyrimidine analogs include 5-fluorodeoxyuridine (floxuridine) and 5-fluorodeoxyuridine monophosphate.

Cytarabine, 4-amino-1β-D-arabinofuranosyl-2 (1H)-pyrimidinone, iscommercially available as CYTOSAR-U® and is commonly known as Ara-C. Itis believed that cytarabine exhibits cell phase specificity at S-phaseby inhibiting DNA chain elongation by terminal incorporation ofcytarabine into the growing DNA chain. Cytarabine is indicated as asingle agent or in combination with other chemotherapy agents in thetreatment of acute leukemia. Other cytidine analogs include5-azacytidine and 2′,2′-difluorodeoxycytidine (gemcitabine).

Mercaptopurine, 1,7-dihydro-6H-purine-6-thione monohydrate, iscommercially available as PURINETHOL®. Mercaptopurine exhibits cellphase specificity at S-phase by inhibiting DNA synthesis by an as of yetunspecified mechanism. Mercaptopurine is indicated as a single agent orin combination with other chemotherapy agents in the treatment of acuteleukemia. A useful mercaptopurine analog is azathioprine.

Thioguanine, 2-amino-1,7-dihydro-6H-purine-6-thione, is commerciallyavailable as TABLOID®. Thioguanine exhibits cell phase specificity atS-phase by inhibiting DNA synthesis by an as of yet unspecifiedmechanism. Thioguanine is indicated as a single agent or in combinationwith other chemotherapy agents in the treatment of acute leukemia. Otherpurine analogs include pentostatin, erythrohydroxynonyladenine,fludarabine phosphate, and cladribine.

Gemcitabine, 2′-deoxy-2′,2′-difluorocytidine monohydrochloride((3-isomer), is commercially available as GEMZAR®. Gemcitabine exhibitscell phase specificity at S-phase and by blocking progression of cellsthrough the G1/S boundary. Gemcitabine is indicated in combination withcisplatin in the treatment of locally advanced non-small cell lungcancer and alone in the treatment of locally advanced pancreatic cancer.

Methotrexate,N-[4[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamicacid, is commercially available as methotrexate sodium. Methotrexateexhibits cell phase effects specifically at S-phase by inhibiting DNAsynthesis, repair and/or replication through the inhibition ofdyhydrofolic acid reductase which is required for synthesis of purinenucleotides and thymidylate. Methotrexate is indicated as a single agentor in combination with other chemotherapy agents in the treatment ofchoriocarcinoma, meningeal leukemia, non-Hodgkin's lymphoma, andcarcinomas of the breast, head, neck, ovary and bladder.

Topoisomerase I inhibitors: Camptothecins, including, camptothecin andcamptothecin derivatives are available or under development asTopoisomerase I inhibitors. Camptothecins cytotoxic activity is believedto be related to its Topoisomerase I inhibitory activity. Examples ofcamptothecins include, but are not limited to irinotecan, topotecan, andthe various optical forms of7-(4-methylpiperazino-methylene)-10,11-ethylenedioxy-20-camptothecindescribed below.

Irinotecan HCl, (4S)-4,11-diethyl-4-hydroxy-9-[(4-piperidinopiperidino)carbonyloxy]-1H-pyrano[3′,4′,6,7]indolizino[1,2-13]quinoline-3,14(4H,12H)-dionehydrochloride, is commercially available as the injectable solutionCAMPTOSAR®. Irinotecan is a derivative of camptothecin which binds,along with its active metabolite SN-38, to the topoisomerase I-DNAcomplex. It is believed that cytotoxicity occurs as a result ofirreparable double strand breaks caused by interaction of thetopoisomerase I:DNA:irintecan or SN-38 ternary complex with replicationenzymes. Irinotecan is indicated for treatment of metastatic cancer ofthe colon or rectum.

Topotecan HCl,(S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3′,4′,6,7]indolizino[1,2-b]quinoline-3,14-(4H,12H)-dionemonohydrochloride, is commercially available as the injectable solutionHYCAMTIN®. Topotecan is a derivative of camptothecin which binds to thetopoisomerase I-DNA complex and prevents religation of singles strandbreaks caused by Topoisomerase I in response to torsional strain of theDNA molecule. Topotecan is indicated for second line treatment ofmetastatic carcinoma of the ovary and small cell lung cancer.

Hormones and hormonal analogues: Hormones and hormonal analogues areuseful compounds for treating cancers in which there is a relationshipbetween the hormone(s) and growth and/or lack of growth of the cancer.Examples of hormones and hormonal analogues useful in cancer treatmentinclude, but are not limited to, adrenocorticosteroids such asprednisone and prednisolone which are useful in the treatment ofmalignant lymphoma and acute leukemia in children; aminoglutethimide andother aromatase inhibitors such as anastrozole, letrazole, vorazole, andexemestane useful in the treatment of adrenocortical carcinoma andhormone dependent breast carcinoma containing estrogen receptors;progestrins such as megestrol acetate useful in the treatment of hormonedependent breast cancer and endometrial carcinoma; estrogens, androgens,and anti-androgens such as flutamide, nilutamide, bicalutamide,cyproterone acetate and 5α-reductases such as finasteride anddutasteride, useful in the treatment of prostatic carcinoma and benignprostatic hypertrophy; anti-estrogens such as tamoxifen, toremifene,raloxifene, droloxifene, iodoxyfene, as well as selective estrogenreceptor modulators (SERMS) such those described in U.S. Pat. Nos.5,681,835, 5,877,219, and 6,207,716, useful in the treatment of hormonedependent breast carcinoma and other susceptible cancers; andgonadotropin-releasing hormone (GnRH) and analogues thereof whichstimulate the release of leutinizing hormone (LH) and/or folliclestimulating hormone (FSH) for the treatment prostatic carcinoma, forinstance, LHRH agonists and antagagonists such as goserelin acetate andluprolide.

Signal transduction pathway inhibitors: Signal transduction pathwayinhibitors are those inhibitors, which block or inhibit a chemicalprocess which evokes an intracellular change. As used herein this changeis cell proliferation or differentiation. Signal tranduction inhibitorsuseful in the present invention include inhibitors of receptor tyrosinekinases, non-receptor tyrosine kinases, SH2/SH3 domain blockers,serine/threonine kinases, phosphotidyl inositol-3 kinases, myo-inositolsignaling, and Ras oncogenes.

Several protein tyrosine kinases catalyse the phosphorylation ofspecific tyrosyl residues in various proteins involved in the regulationof cell growth. Such protein tyrosine kinases can be broadly classifiedas receptor or non-receptor kinases.

Receptor tyrosine kinases are transmembrane proteins having anextracellular ligand binding domain, a transmembrane domain, and atyrosine kinase domain. Receptor tyrosine kinases are involved in theregulation of cell growth and are generally termed growth factorreceptors. Inappropriate or uncontrolled activation of many of thesekinases, i.e. aberrant kinase growth factor receptor activity, forexample by overexpression or mutation, has been shown to result inuncontrolled cell growth. Accordingly, the aberrant activity of suchkinases has been linked to malignant tissue growth. Consequently,inhibitors of such kinases could provide cancer treatment methods.Growth factor receptors include, for example, epidermal growth factorreceptor (EGFr), platelet derived growth factor receptor (PDGFr), erbB2,erbB4, ret, vascular endothelial growth factor receptor (VEGFr),tyrosine kinase with immunoglobulin-like and epidermal growth factorhomology domains (TIE-2), insulin growth factor-I (IGFI) receptor,macrophage colony stimulating factor (cfms), BTK, ckit, cmet, fibroblastgrowth factor (FGF) receptors, Trk receptors (TrkA, TrkB, and TrkC),ephrin (eph) receptors, and the RET protooncogene. Several inhibitors ofgrowth receptors are under development and include ligand antagonists,antibodies, tyrosine kinase inhibitors and anti-sense oligonucleotides.Growth factor receptors and agents that inhibit growth factor receptorfunction are described, for instance, in Kath, John C., Exp. Opin. Ther.Patents (2000) 10(6):803-818; Shawver et al DDT Vol 2, No. 2 Feb. 1997;and Lofts, F. J. et al, “Growth factor receptors as targets,” NewMolecular Targets for Cancer Chemotherapy, ed. Workman, Paul and Kerr,David, CRC press 1994, London.

Tyrosine kinases, which are not growth factor receptor kinases aretermed non-receptor tyrosine kinases. Non-receptor tyrosine kinasesuseful in the present invention, which are targets or potential targetsof anti-cancer drugs, include cSrc, Lck, Fyn, Yes, Jak, cAbl, FAK (Focaladhesion kinase), Brutons tyrosine kinase, and Bcr-Abl. Suchnon-receptor kinases and agents which inhibit non-receptor tyrosinekinase function are described in Sinh, S. and Corey, S. J., (1999)Journal of Hematotherapy and Stem Cell Research 8 (5): 465-80; andBolen, J. B., Brugge, J. S., (1997) Annual review of Immunology. 15:371-404.

SH2/SH3 domain blockers are agents that disrupt SH2 or SH3 domainbinding in a variety of enzymes or adaptor proteins including, PI3-K p85subunit, Src family kinases, adaptor molecules (Shc, Crk, Nck, Grb2) andRas-GAP. SH2/SH3 domains as targets for anti-cancer drugs are discussedin Smithgall, T. E. (1995), Journal of Pharmacological and ToxicologicalMethods. 34(3) 125-32.

Inhibitors of Serine/Threonine Kinases including MAP kinase cascadeblockers which include blockers of Raf kinases (rafk), Mitogen orExtracellular Regulated Kinase (MEKs), and Extracellular RegulatedKinases (ERKs); and Protein kinase C family member blockers includingblockers of PKCs (alpha, beta, gamma, epsilon, mu, lambda, iota, zeta).IkB kinase family (IKKa, IKKb), PKB family kinases, akt kinase familymembers, and TGF beta receptor kinases. Such Serine/Threonine kinasesand inhibitors thereof are described in Yamamoto, T., Taya, S.,Kaibuchi, K., (1999), Journal of Biochemistry. 126 (5) 799-803; Brodt,P, Samani, A., and Navab, R. (2000), Biochemical Pharmacology, 60.1101-1107; Massague, J., Weis-Garcia, F. (1996) Cancer Surveys.27:41-64; Philip, P. A., and Harris, A. L. (1995), Cancer Treatment andResearch. 78: 3-27, Lackey, K. et al Bioorganic and Medicinal ChemistryLetters, (10), 2000, 223-226; U.S. Pat. No. 6,268,391; andMartinez-lacaci, L., et al, Int. J. Cancer (2000), 88(1), 44-52.

Inhibitors of Phosphotidyl inositol-3 Kinase family members includingblockers of PI3-kinase, ATM, DNA-PK, and Ku are also useful in thepresent invention. Such kinases are discussed in Abraham, R. T. (1996),Current Opinion in Immunology. 8 (3) 412-8; Canman, C. E., Lim, D. S.(1998), Oncogene 17 (25) 3301-3308; Jackson, S. P. (1997), InternationalJournal of Biochemistry and Cell Biology. 29 (7):935-8; and Zhong, H. etal, Cancer res, (2000) 60(6), 1541-1545.

Also useful in the present invention are Myo-inositol signalinginhibitors such as phospholipase C blockers and Myoinositol analogues.Such signal inhibitors are described in Powis, G., and Kozikowski A.,(1994) New Molecular Targets for Cancer Chemotherapy ed., Paul Workmanand David Kerr, CRC press 1994, London.

Another group of signal transduction pathway inhibitors are inhibitorsof Ras Oncogene. Such inhibitors include inhibitors offarnesyltransferase, geranyl-geranyl transferase, and CAAX proteases aswell as anti-sense oligonucleotides, ribozymes and immunotherapy. Suchinhibitors have been shown to block ras activation in cells containingwild type mutant ras, thereby acting as antiproliferation agents. Rasoncogene inhibition is discussed in Scharovsky, O. G., Rozados, V. R.,Gervasoni, S. I. Matar, P. (2000), Journal of Biomedical Science. 7(4)292-8; Ashby, M. N. (1998), Current Opinion in Lipidology. 9 (2) 99-102;and BioChim. Biophys. Acta, (19899) 1423(3):19-30.

As mentioned above, antibody antagonists to receptor kinase ligandbinding may also serve as signal transduction inhibitors. This group ofsignal transduction pathway inhibitors includes the use of humanizedantibodies to the extracellular ligand binding domain of receptortyrosine kinases. For example Imclone C225 EGFR specific antibody (seeGreen, M. C. et al, Monoclonal Antibody Therapy for Solid Tumors, CancerTreat. Rev., (2000), 26(4), 269-286); Herceptin erbB2 antibody (seeTyrosine Kinase Signalling in Breast cancer:erbB Family ReceptorTyrosine Kinases, Breast cancer Res., 2000, 2(3), 176-183); and 2CBVEGFR2 specific antibody (see Brekken, R. A. et al, Selective Inhibitionof VEGFR2 Activity by a monoclonal Anti-VEGF antibody blocks tumorgrowth in mice, Cancer Res. (2000) 60, 5117-5124).

Anti-angiogenic agents: Anti-angiogenic agents including non-receptorangiogenesis inhibitors may also be useful. Anti-angiogenic agents suchas those which inhibit the effects of vascular edothelial growth factor,(for example the anti-vascular endothelial cell growth factor antibodybevacizumab [Avastin™], and compounds that work by other mechanisms (forexample linomide, inhibitors of integrin αvβ3 function, endostatin andangiostatin);

Immunotherapeutic agents: Agents used in immunotherapeutic regimens mayalso be useful in combination with the compounds of formula (I).Immunotherapy approaches, including for example ex-vivo and in-vivoapproaches to increase the immunogenecity of patient tumour cells, suchas transfection with cytokines such as interleukin 2, interleukin 4 orgranulocyte-macrophage colony stimulating factor, approaches to decreaseT-cell anergy, approaches using transfected immune cells such ascytokine-transfected dendritic cells, approaches usingcytokine-transfected tumour cell lines and approaches usinganti-idiotypic antibodies

Proapoptotoc agents: Agents used in proapoptotic regimens (e.g., bcl-2antisense oligonucleotides) may also be used in the combination of thepresent invention.

Cell cycle signalling inhibitors: Cell cycle signalling inhibitorsinhibit molecules involved in the control of the cell cycle. A family ofprotein kinases called cyclin dependent kinases (CDKs) and theirinteraction with a family of proteins termed cyclins controlsprogression through the eukaryotic cell cycle. The coordinate activationand inactivation of different cyclin/CDK complexes is necessary fornormal progression through the cell cycle. Several inhibitors of cellcycle signalling are under development. For instance, examples of cyclindependent kinases, including CDK2, CDK4, and CDK6 and inhibitors for thesame are described in, for instance, Rosania et al, Exp. Opin. Ther.Patents (2000) 10(2):215-230.

In one embodiment, the combination of the present invention comprises acompound of formula I or a salt or solvate thereof and at least oneanti-neoplastic agent selected from anti-microtubule agents, platinumcoordination complexes, alkylating agents, antibiotic agents,topoisomerase II inhibitors, antimetabolites, topoisomerase Iinhibitors, hormones and hormonal analogues, signal transduction pathwayinhibitors, non-receptor tyrosine angiogenesis inhibitors,immunotherapeutic agents, proapoptotic agents, and cell cycle signalinginhibitors.

In one embodiment, the combination of the present invention comprises acompound of formula I or a salt or solvate thereof and at least oneanti-neoplastic agent which is an anti-microtubule agent selected fromditerpenoids and vinca alkaloids.

In a further embodiment, the at least one anti-neoplastic agent is aditerpenoid.

In a further embodiment, the at least one anti-neoplastic agent is avinca alkaloid.

In one embodiment, the combination of the present invention comprises acompound of formula I or a salt or solvate thereof and at least oneanti-neoplastic agent, which is a platinum coordination complex.

In a further embodiment, the at least one anti-neoplastic agent ispaclitaxel, carboplatin, or vinorelbine.

In a further embodiment, the at least one anti-neoplastic agent iscarboplatin.

In a further embodiment, the at least one anti-neoplastic agent isvinorelbine.

In a further embodiment, the at least one anti-neoplastic agent ispaclitaxel.

In one embodiment, the combination of the present invention comprises acompound of formula I and salts or solvates thereof and at least oneanti-neoplastic agent which is a signal transduction pathway inhibitor.

In a further embodiment the signal transduction pathway inhibitor is aninhibitor of a growth factor receptor kinase VEGFR2, TIE2, PDGFR, BTK,erbB2, EGFr, IGFR-1, TrkA, TrkB, TrkC, or c-fms.

In a further embodiment the signal transduction pathway inhibitor is aninhibitor of a serine/threonine kinase rafk, akt, or PKC-zeta.

In a further embodiment the signal transduction pathway inhibitor is aninhibitor of a non-receptor tyrosine kinase selected from the src familyof kinases.

In a further embodiment the signal transduction pathway inhibitor is aninhibitor of c-src.

In a further embodiment the signal transduction pathway inhibitor is aninhibitor of Ras oncogene selected from inhibitors of farnesyltransferase and geranylgeranyl transferase.

In a further embodiment the signal transduction pathway inhibitor is aninhibitor of a serine/threonine kinase selected from the groupconsisting of PI3K.

In a further embodiment the signal transduction pathway inhibitor is adual EGFr/erbB2 inhibitor, for exampleN-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine(structure below):

In one embodiment, the combination of the present invention comprises acompound of formula I or a salt or solvate thereof and at least oneanti-neoplastic agent which is a cell cycle signaling inhibitor.

In further embodiment, cell cycle signaling inhibitor is an inhibitor ofCDK2, CDK4 or CDK6.

In one embodiment the mammal in the methods and uses of the presentinvention is a human.

As indicated, therapeutically effective amounts of the active componentsin combinations and combination therapies of the invention (a PD-1antagonist and one or both of Compound A and Compound B) areadministered to a human. Typically, the therapeutically effective amountof the administered agents of the present invention will depend upon anumber of factors including, for example, the age and weight of thesubject, the precise condition requiring treatment, the severity of thecondition, the nature of the formulation, and the route ofadministration. Ultimately, the therapeutically effective amount will beat the discretion of the attendant physician.

The combinations of the present invention are tested for efficacy,advantageous and synergistic properties according to known procedures.Suitably, the combinations of the invention are tested for efficacy,advantageous and synergistic properties generally according to thefollowing combination cell proliferation assays. Cells are plated in384-well plates at 500 cells/well in culture media appropriate for eachcell type, supplemented with 10% FBS and 1% penicillin/streptomycin, andincubated overnight at 37° C., 5% CO₂. Cells are treated in a gridmanner with dilution of Compound A (20 dilutions, including no compound,of 2-fold dilutions starting from 1-20 mM depending of compound) fromleft to right on 384-well plate; and also optionally treated withCompound B (20 dilutions, including no compound, of 2-fold dilutionsstarting from 1-20 mM depending of compound) from top to bottom on384-well plate; and also treated with an anti-PD-1 antibody or antigenbinding fragment thereof and incubated as above for a further 72 hours.In some instances compounds are added in a staggered manner andincubation time can be extended up to 7 days. Cell growth is measuredusing CellTiter-Glo® reagent according to the manufacturer's protocoland signals are read on a PerkinElmer EnVision™ reader set forluminescence mode with a 0.5-second read. Data are analyzed as describedbelow.

Results are expressed as a percentage of the t=0 value and plottedagainst compound(s) concentration. The t=0 value is normalized to 100%and represents the number of cells present at the time of compoundaddition. The cellular response is determined for each compound and/orcompound combination using a 4- or 6-parameter curve fit of cellviability against concentration using the IDBS XLfit plug-in forMicrosoft Excel software and determining the concentration required for50% inhibition of cell growth (gIC₅₀). Background correction is made bysubtraction of values from wells containing no cells. For each drugcombination a Combination Index (CI), Excess Over Highest Single Agent(EOHSA) and Excess Over Bliss (EOBliss) are calculated according toknown methods such as described in Chou and Talalay (1984) Advances inEnzyme Regulation, 22, 37 to 55; and Berenbaum, M C (1981) Adv. CancerResearch, 35, 269-335.

The combinations of the present invention are tested in the above assaysto determine advantageous therapeutic utility in treating cancer.

The following examples are intended for illustration only and are notintended to limit the scope of the invention in any way.

Example 1 Kit Composition

The sucrose, microcrystalline cellulose and the compounds A and B of theinvented combination, as shown in Tables I and II below, areindividually mixed and granulated in the proportions shown with a 10%gelatin solution. The wet granules are screened, dried, mixed with thestarch, talc and stearic acid, then screened and compressed into atablet. A vial of MK-3475 is also included in the kit as described inTable III.

TABLE I INGREDIENTS AMOUNTSN-{3-[3-cyclopropyl-5-(2-fluoro-4-iodo-phenylamino)6,8- 2 mgdimethyl-2,4,7-trioxo-3,4,6,7-tetrahydro-2H-pyrido[4,3-d]pyrimidin-1-yl]phenyl}acetamide dimethyl sulfoxide (the dimethylsulfoxide solvate of Compound A) Microcrystalline cellulose 300 mgSucrose 4 mg Starch 2 mg Talc 1 mg stearic acid 0.5 mg

TABLE II INGREDIENTSN-{3-[5-(2-Amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3- 200 mgthiazol-4-yl]-2-fluorophenyl}-2,6- difluorobenzenesulfonamidemethanesulfonate, (the methanesulfonate salt of Compound B)Microcrystalline cellulose 200 mg Sucrose 10 mg Starch 40 mg Talc 20 mgstearic acid 5 mg

TABLE III MK-3475 may be supplied as a solution consisting of 25 mg/mlMK-3475, 7% (w/v) Sucrose, 0.02% (w/v) polysorbate 80 in 10 mM histidinebuffer pH 5.5.

Example 2 Anti-Tumor Response of Concurrent Administration of a PD-1Antagonist and Trametinib to Tumor-Bearing Mice

This experiment compared the anti-tumor response of tumor-bearing miceto treatment with one of three regimens: (i) monotherapy with a murineanti-mouse PD-1 monoclonal antibody of murine isotype IgG1 (Anti-PD1);(ii) monotherapy with trametinib and (iii) combination therapy withanti-PD1 and trametinib administered concurrently. Murine isotype IgG1is the murine counterpart isotype to human isotype IgG4.

While human tumor cells or tumor explants can be grown inimmunodeficient animals as xenografts, they cannot be used for testingimmunotherapeutics because of the lack of a functional immune system.For a meaningful evaluation of immunotherapeutics or combination ofimmunotherapeutics with other agents, it is necessary to use a syngeneicmodel in which syngeneic tumors are grown in animals with an intactimmune system. CT-26 is a murine colorectal adenocarcinoma cell linesyngeneic to the BALB/c mouse strain. CT-26 carries a KRAS mutation thatactivates the mitogen-activating protein kinase pathway, which makes ita relevant model to evaluate sensitivity to trametinib (an inhibitor ofmitogen-activated protein kinase kinase). This is also a relevant modelsystem for evaluating the mechanism of action for an anti-PD-1 antibodybecause of the translatable molecular profile of this tumorpost-anti-PD-1 therapy.

Tumor-bearing mice for this study were initiated by implanting 3×10⁵log-phase and sub-confluent CT-26 cells on the right lower dorsal flankof 7-8 week old female BALB/c mice with an average body weight of 20grams. When the mean tumor volume in these mice reached ˜126 cubic mm(FIG. 8B, left panel marked Day 0), the tumor-bearing mice wererandomized to 4 treatment groups of 12 mice per group: (1)Isotype+Vehicle control group; (2) Anti-PD1+Vehicle control; (3)trametinib+Isotype control and (4) Anti-PD1+trametinib. The Vehiclecontrol was 0.5% HPMC (Hydroxypropylmethylcellulose, Sigma) and 0.2%Tween 80 (Sigma) in injection-grade water pH 8.0. The Isotype controlwas a mouse monoclonal antibody specific for adenoviral hexon 25 and wasof the murine isotype IgG1. Anti-PD1 was administered to treatmentgroups 2 and 4 at 10 mg/kg i.p., every 5 days for each of 5 cycles, withthe first dosing designated as Day 0 in FIG. 8. Trametinib wasadministered to treatment groups 3 and 4 at 1 mg/kg every day for 23days, with the first dosing designated as Day 0 in FIG. 8.

Surprisingly, administration of trametinib, a potential T cellimmunosuppressive agent, did not appear to antagonize the anti-tumoreffects of Anti-PD1 in the combination treatment arm. In contrast, asdemonstrated by the results shown in FIG. 8, the mean anti-tumorresponse of concurrent administration of Anti-PD1 and trametinib wasgreater (p<0.001) than the anti-tumor response observed with eithersingle agent treatment. In addition, the combination therapy resulted ina complete regression (CR) (no measurable tumor) in 17% of the cohort (2out of 12 animals) at the end of the 23 day treatment period, while noCRs were observed in the single agent treatment groups. Comparing meantumor volumes at the end of the study (FIG. 8B, right panel) using oneway ANOVA/Bonferroni, the tumor volumes of mice treated with thecombination of trametinib+Anti-PD1 were significantly smaller than thosetreated with trametinib alone.

Example 3 Clinical Safety and Efficacy of a Combination of a PD-1Antagonist, a MEK Inhibitor and a BRAF Inhibitor

A Phase I/II Study is performed to Assess the Safety and Efficacy ofMK-3475 in Combination with Trametinib and Dabrafenib in Subjects withAdvanced Melanoma. This study is a multi-center, worldwide, Phase I/II3-part trial of intravenous (IV) MK-3475 in combination with oraldabrafenib and/or trametinib in subjects with advanced or metastaticmelanoma to be conducted in conformance with Good Clinical Practices.

Part 1 is a nonrandomized, multi-site, open-label portion of the studyusing a traditional 3+3 design for dose escalation to evaluate safety,tolerability, and dosing of MK-3475 in combination with dabrafenib andtrametinib (MK+D+T) in BRAF mutation-positive (V600 E/K) melanomasubjects. Part 2 is a nonrandomized, multi-site, open-label portion ofthe study using expansion cohort(s) to further evaluate safety andconfirm dose of (MK+D+T). Also in Part 2, expansion cohort(s) will beused to further evaluate safety and preliminary efficacy in the (MK+T)combination. Part 3 is a randomized (1:1), active-controlled,multi-site, two-arm study of the confirmed dose of the tripletcombination (MK+D+T) versus placebo in combination with D+T (PBO+D+T).Subjects will be stratified by Eastern Cooperative Oncology Group (ECOG)Performance Scale (0 vs. 1) and Lactate Dehydrogenase (LDH) level(>1.1×ULN vs. ≦1.1×ULN). A more detailed summary of the study ispresented in the table below.

Clinical Indication The treatment of subjects with advanced ormetastatic melanoma Trial Type Interventional Type of control Notreatment control in Parts 1 and 2. Placebo (standard, active therapywith a placebo add-on) in Part 3. Route of administration Intravenous(MK-3475) Oral (trametinib) Oral (dabrafenib) Trial Blinding Parts 1 and2: Unblinded Open Label Part 3: Double-blind Treatment Groups In Part 1(3 + 3 design), cohorts of 3 or 6 subjects with BRAF mutant [V600 E/K]melanoma will receive escalating doses of MK-3475 in combination withtrametinib 2 mg QD and dabrafenib 150 mg BID: MK-3475 2 mg/kg q3 weeksMK-3475 10 mg/kg q2 weeks Additionally in Part 1 (3 + 3 design), cohortsof 3 or 6 subjects with BRAF wild type [without V600] melanoma) willreceive escalating doses of MK-3475 in combination with trametinib 2 mgQD: MK-3475 2 mg/kg q3 weeks MK-3475 10 mg/kg q2 weeks Approximately 18subjects will be enrolled in Part 1 (~9 for the (MK + D + T) combinationtherapy and ~9 for the (MK + T) combination therapy). In Part 1, andonly in the event dose level 1 is not tolerable in the (MK + D + T)combination therapy, cohorts of 3 or 6 subjects with BRAF mutant [V600E/K] melanoma will receive escalating doses of MK-3475 in combinationwith dabrafenib 150 mg BID (3 + 3 design): MK-3475 2 mg/kg q3 weeksMK-3475 10 mg/kg q2 weeks Approximately 9 subjects will be enrolled forthe (MK + D) combination therapy, but only if dose level 1 is nottolerable in the (MK + D + T) combination. Part 2 will expand cohort(s)from Part 1 for dose confirmation. Approximately 66 subjects will beenrolled in Part 2, ~20 for (MK + D + T) and ~46 for (MK + T). In Part3, approximately 120 subjects with BRAF mutant [V600 E/K] melanoma willbe randomized 1:1 to either: (a) the confirmed dose of MK-3475,trametinib 2 mg QD, dabrafenib 150 mg BID, or, (b) placebo (saline IV),trametinib 2 mg QD, dabrafenib 150 mg BID. Note: The triplet dosecombination of all treatments used in Part 3 will be confirmed by Parts1 and 2 of the study. Number of trial subjects Approximately 89 subjectswill be enrolled in the investigation of the (MK + D + T) combinationtherapy (this total includes subjects from specific arms in Parts 1 and2, and subjects in the blinded active (MK + D + T) arm from Part 3).Additionally, 60 subjects will receive PBO (saline IV) + (D + T) in Part3 of the study. ~55 subjects will be enrolled in the investigation ofthe (MK + T) combination therapy (Parts 1 and 2 only). In the overallstudy, ~204 subjects will be enrolled. Estimated duration of trial Thesponsor estimates that the trial will require approximately 44 monthsfrom the time the first subject signs the informed consent until thelast subject's last visit. Duration of Participation Each subject willparticipate in the trial from the time the subject signs the InformedConsent Form (ICF) through the final protocol-specified contact. After ascreening phase of up to 28 days, eligible subjects will receiveassigned treatment on Day 1 of the dosing cycle. Treatment with MK-3475,trametinib, and/or dabrafenib will continue until documented diseaseprogression, unacceptable adverse event(s), intercurrent illness thatprevents further administration of treatment, investigator's decision towithdraw the subject, subject withdraws consent, pregnancy of thesubject, noncompliance with trial treatment or procedure requirements,or administrative reasons. MK-3475 treated subjects who attain acomplete response after at least 6 months of study treatment mayconsider stopping MK-3475 treatment (at the discretion of theinvestigator after receiving at least two doses beyond the initialdetermination of complete response), while continuing on treatment withtrametinib and/or dabrafenib. Subjects who stop MK- 3475 may be eligiblefor re-treatment after experiencing disease progression at thediscretion of the investigator if they meet the criteria forre-treatment; this will be designated the Second Course Phase. MK-3475treatment may continue for a maximum of 24 months. After the end of alltreatments, each subject will be followed for a minimum of 30 days foradverse event monitoring (serious adverse events will be collected forup to 90 days after the end of treatment). Subjects will havepost-treatment follow-up for disease status, including initiating anon-study cancer treatment and experiencing disease progression, untildeath, withdrawing consent, or becoming lost to follow-up. RandomizationRatio Part 3: 1:1The table below provides a brief description of the sequences in thesequence listing.

SEQ ID NO: Description 1 hPD-1.08A light chain CDR1 2 hPD-1.08A lightchain CDR2 3 hPD-1-08A light chain CDR3 4 hPD-1.08A heavy chain CDR1 5hPD-1.08A heavy chain CDR2 6 hPD-1.08A heavy chain CDR3 7 hPD-1.09Alight chain CDR1 8 hPD-1.09A light chain CDR2 9 hPD-1.09A light chainCDR3 10 hPD-1.09A heavy chain CDR1 11 hPD-1.09A heavy chain CDR2 12hPD-1.09A heavy chain CDR3 13 109A-H heavy chain variable region 14409A-H heavy chain full length 15 K09A-L-11 light chain variable region16 K09A-L-16 light chain variable region 17 K09A-L-17 light chainvariable region 18 K09A-L-11 light chain full length 19 K09A-L-16 lightchain full length 20 K09A-L-17 light chain full length 21 MK-3475 Heavychain 22 MK-3475 Light chain 23 Nivolumab Heavy chain 24 Nivolumab lightchain

While the preferred embodiments of the invention are illustrated by theabove, it is to be understood that the invention is not limited to theprecise instructions herein disclosed and that the right to allmodifications coming within the scope of the following claims isreserved.

1. A combination comprising a PD-1 antagonist and one or both of aCompound A and a Compound B, wherein: the Compound B is a compound ofstructure (II)

or a pharmaceutically acceptable salt thereof; and the Compound A is acompound of structure (I):

or a pharmaceutically acceptable salt or solvate thereof.
 2. Acombination comprising a PD-1 antagonist and one or both of a Compound Aand a Compound B, wherein the Compound B isN-{3-[5-(2-Amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamidemethanesulfonate; and the Compound A isN-{3-[3-cyclopropyl-5-(2-fluoro-4-iodo-phenylamino)6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydro-2H-pyrido[4,3-d]pyrimidin-1-yl]phenyl}acetamidedimethyl sulfoxide (solvate).
 3. (canceled)
 4. The combination of claim1, wherein the Compound A is trametinib, the Compound B is dabrafeniband the PD-1 antagonist is an anti-human PD-1 antibody.
 5. Thecombination of claim 4, wherein the anti-human PD-1 antibody is MK-3475.6. A pharmaceutical composition comprising a PD-1 antagonist, whereinthe pharmaceutical composition comprises one or both of a Compound A anda Compound B together with a pharmaceutically acceptable diluent orcarrier, and wherein: the Compound A is a compound of structure (I):

or a pharmaceutically acceptable salt or solvate thereof; and thecompound B is a compound of structure (II):

or a pharmaceutically acceptable salt thereof.
 7. The composition ofclaim 6, wherein: the composition comprises each of Compound A andCompound B; and the PD-1 antagonist is an anti-human PD-1 antibody or anantigen binding fragment thereof.
 8. The composition of claim 6,wherein: the composition does not comprise Compound B; and the PD-1antagonist is an anti-human PD-1 antibody or an antigen binding fragmentthereof.
 9. The composition of claim 6, wherein: the composition doesnot comprise Compound A; and the PD-1 antagonist is an anti-human PD-1antibody or an antigen binding fragment thereof.
 10. A pharmaceuticalcomposition comprising an anti-human PD-1 antibody or an antigen bindingfragment thereof together with a pharmaceutically acceptable diluent orcarrier, wherein the combination therapy comprises the pharmaceuticalcomposition and one or both of a Compound A and a Compound B, whereinthe Compound A is a compound of structure (I):

or a pharmaceutically acceptable salt or solvate thereof; and whereinthe Compound B is a compound of structure (II):

or a pharmaceutically acceptable salt thereof.
 11. The composition ofclaim 9, wherein the anti-human PD-1 antibody is MK-3475 or nivolumab,and the combination therapy comprises each of Compound A and Compound B.12. The composition of claim 10, wherein the anti-human PD-1 antibody isMK-3475 or nivolumab, and the combination therapy does not compriseCompound B.
 13. The composition of claim 10, wherein the anti-human PD-1antibody is MK-3475 or nivolumab, and the composition does not compriseCompound A.
 14. The composition of claim 10, wherein the Compound A istrametinib, the Compound B is dabrafenib, the anti-human PD-1 antibodyis MK-3475.
 15. (canceled)
 16. A method of treating cancer in a mammalcomprising administering to said mammal a combination therapy, whereinthe combination therapy comprises a therapeutically effective amount ofa PD-1 antagonist and one or both of a therapeutically effective amountof a Compound A and a therapeutically effective amount of a Compound B,wherein: the Compound B is a compound of structure (II):

or a pharmaceutically acceptable salt thereof; and the Compound A is acompound of structure (I):

or a pharmaceutically acceptable salt or solvate thereof.
 17. The methodof claim 16, wherein the mammal is a human, the PD-1 antagonist is ananti-human PD-1 antibody or an antigen binding fragment thereof, theCompound B isN-{3-[5-(2-Amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamideor a pharmaceutically acceptable salt thereof and the Compound A isN-{3-[3-cyclopropyl-5-(2-fluoro-4-iodo-phenylamino)6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydro-2H-pyrido[4,3-d]pyrimidin-1-yl]phenyl}acetamide,or a pharmaceutically acceptable salt or solvate thereof.
 18. The methodof claim 16, wherein the mammal is a human, the PD-1 antagonist isMK-3475, the Compound B isN-{3-[5-(2-Amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamidemethanesulfonate, and the Compound A isN-{3-[3-cyclopropyl-5-(2-fluoro-4-iodo-phenylamino)6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydro-2H-pyrido[4,3-d]pyrimidin-1-yl]phenyl}acetamidedimethyl sulfoxide solvate.
 19. (canceled)
 20. The method of claim 16,wherein the human has been diagnosed with advanced melanoma that testspositive for a BRAF V600 mutation and the combination therapy comprisestrametinib, dabrafenib and MK-3475.
 21. The method of claim 20, whereinthe therapeutically effective amount of MK-3475 is 2 mg/kg once every 3weeks or 10 mg/kg once every 2 weeks, the therapeutically effectiveamount of trametinib is 2 mg once daily and the therapeuticallyeffective amount of dabrafenib is 150 mg twice daily.
 22. The method ofclaim 16, wherein the human has been diagnosed with advanced melanomathat tests negative for a BRAF V600 mutation and wherein the combinationtherapy does not comprise the Compound B.
 23. The method of claim 22,wherein the therapeutically effective amount of MK-3475 is 2 mg/kg onceevery 3 weeks or 10 mg/kg once every 2 weeks and the therapeuticallyeffective amount of trametinib is 2 mg once daily.
 24. The method ofclaim 16, wherein the human has been diagnosed with advanced melanomathat tests positive for a BRAF V600 mutation and the combination therapydoes not comprise Compound A.
 25. The method of claim 24, wherein thetherapeutically effective amount of MK-3475 is 2 mg/kg once every 3weeks or 10 mg/kg once every 2 weeks and the therapeutically effectiveamount of dabrafenib is 150 mg twice daily.